WO2023070317A1 - Bifunctional nanobody based on dc, and construction method therefor and use thereof - Google Patents

Bifunctional nanobody based on dc, and construction method therefor and use thereof Download PDF

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WO2023070317A1
WO2023070317A1 PCT/CN2021/126429 CN2021126429W WO2023070317A1 WO 2023070317 A1 WO2023070317 A1 WO 2023070317A1 CN 2021126429 W CN2021126429 W CN 2021126429W WO 2023070317 A1 WO2023070317 A1 WO 2023070317A1
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porcine
nanobody
bifunctional
gene
targeting
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French (fr)
Chinese (zh)
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程海卫
侯立婷
杜露平
于晓明
乔绪稳
张元鹏
李兰
王义伟
张浩明
秦竹
杨利
陈瑾
郑其升
侯继波
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江苏省农业科学院
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Priority to PCT/CN2021/126429 priority Critical patent/WO2023070317A1/en
Priority to CN202210477668.XA priority patent/CN115304678A/en
Priority to GB2212254.3A priority patent/GB2612413A/en
Publication of WO2023070317A1 publication Critical patent/WO2023070317A1/en

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    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
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    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a DC cell-based bifunctional nanobody and its construction method and application.
  • DC Dendritic cells
  • the active targeted delivery of antigens to DC cells is of great significance for improving the efficiency of antigen presentation and improving the efficacy of antigen immunity.
  • the DC targeting of antigens is mainly achieved by targeting DC cell surface receptors. After exogenous antigens are targeted to DC cells by specific antibodies or ligands, DC cells absorb, process and present them. Following the MHC II or cross-presentation pathway, it is assembled with MHC molecules to form a peptide-MHC complex, which is transported to the cell surface and presented to the corresponding effector cells to activate the immune response.
  • targeted antigens Compared with non-targeted antigens, targeted antigens have significantly improved antigen presentation efficiency and reduced antigen dosage, and can even induce CD8 + T cell immune responses through cross-presentation pathways, which are useful in antiviral infection and tumor treatment. It has good application value, and several DC-targeted human vaccines have entered the clinical trial stage.
  • CD205 belongs to the macrophage mannose receptor family in C-type lectin receptors (CLRs), also known as DEC-205 or Ly75, which is in Widely distributed in tissues, highly expressed in DC cells and thymus epithelial cells, rarely expressed or hardly expressed in B cells, T cells, NK cells and macrophages, CD205 is currently the DC cell in the T cell region of the body's lymphoid organs The most widely expressed unique receptor in , which plays an important role in antigen presentation.
  • CLRs C-type lectin receptors
  • CD205 targeting can increase the presentation efficiency of OVA antigens by at least 100 times, and reduce the amount of antigens by nearly 1000 times; by targeting HBV preS antigens to CD205, it can produce Efficient IgG1 and IgG2a antibody responses have preventive and therapeutic effects on hepatitis B virus infection; targeting dengue virus NS1 antigen and EDIII antigen to CD205 can stimulate efficient antibody responses and T cell responses in mice, resulting in better In human melanoma clinical trials, it was found that by targeting NY-ESO-1 antigen to CD205, highly efficient humoral and cellular immune responses could be generated, and some patients showed stable symptoms and tumors Regression and shrinkage of lesions.
  • CD205 targeting can reduce the amount of antigen used, induce efficient immune response, and has good development potential in antiviral infection and tumor therapy.
  • CD205 has become an important target in the development and application of new vaccine preparations.
  • CD205 targeting The vaccine for human use has also entered the stage of clinical trials.
  • IFN- ⁇ levels and antibody levels can be significantly increased; by targeting avian influenza virus HA antigen to chicken CD205, highly effective antibody levels can be produced 14 days after immunization ;By targeting the Rift Valley fever virus eGn antigen to sheep CD205, although IFN- ⁇ levels can be increased, antibody levels and challenge protection are reduced; by targeting the GP345M fusion antigen of PRRSV to pig CD205, although it can produce Better IFN- ⁇ level and antibody level, but can not produce challenge protection, and the difference in lung lesions of pigs is not significant; CD205 targeting FMDV, PEDV studies have not been reported.
  • the DC targeting of antigens mainly uses specific monoclonal antibodies or single-chain antibodies corresponding to DC surface receptors to assemble with protein antigens through chemical coupling or gene fusion expression. This process is cumbersome and easily affects the antigen structure. Therefore, exploring new antigen assembly strategies is of great significance for improving the efficacy of DC-targeted immunity.
  • Nanobodies are the smallest antigen-binding fragments known to have complete functions. Compared with monoclonal antibodies and single-chain antibodies, nanobodies have a relatively small molecular weight (about 15KDa), low immunogenicity, high stability, and easy utilization of microbial genes. With the characteristics of high-efficiency production of engineering systems, it has good application prospects in the development of cheap and efficient therapeutic antibodies and detection reagents.
  • the multivalent and multispecific nanobodies transformed by genetic engineering solve the problem of poor serum stability of monovalent nanobodies and problems in vivo. Short half-life and other shortcomings, and at the same time, different targets can be used to synergistically promote the immune response, which is considered by many authoritative research institutions as a potential drug for the treatment of cancer.
  • the technical problem to be solved by the present invention is to provide a bifunctional nanobody that can be used for antigen-targeting porcine DC cells.
  • the present invention adopts a new technical idea, utilizes the specific combination between the antigen and the nanobody and the bridging effect of the bifunctional nanobody to realize the DC targeting of the antigen, and this method can avoid chemical coupling and gene fusion expression methods
  • the complete pathogenic particle can be used to replace the conventional protein antigen, and the natural epitope of the complete pathogenic particle can be fully utilized, which is easy to be applied in clinical practice.
  • the technical problem to be solved in the present invention is to provide a method for constructing the bifunctional nanobody.
  • the final technical problem to be solved by the present invention is to provide the application of the bifunctional nanobody.
  • the present invention provides a DC cell-based bifunctional nanobody.
  • the bifunctional nanobody uses a linker element to transfer the gene of a DC cell-based target protein-specific nanobody to The target gene fragment is obtained after being connected with the gene encoding the virus antigen-specific nanobody with a particle size below 150nm, and the obtained target protein is recombinantly expressed.
  • the DC cell-based target protein-specific Nanobody includes a Nanobody encoding DC cell specificity or a Nanobody encoding DC cell antigen-presenting receptor specificity.
  • the target protein-specific nanobody of the porcine DC cells and the porcine virus antigen-specific nanobody with a particle size within 150 nm are screened separately by using phage display technology.
  • the particle size of the porcine virus antigen is 17nm-130nm.
  • said porcine virus antigen comprises porcine circovirus antigen (diameter of virus particle is about 17nm), porcine parvovirus antigen (diameter of virus particle is about 20nm), porcine foot-and-mouth disease virus antigen (diameter of virus particle is about 25nm), classical swine fever
  • porcine circovirus antigen (diameter of virus particle is about 17nm)
  • porcine parvovirus antigen diameter of virus particle is about 20nm
  • porcine foot-and-mouth disease virus antigen (diameter of virus particle is about 25nm)
  • classical swine fever One or more of viral antigens (virion diameter is about 50nm), porcine reproductive and respiratory syndrome virus antigen (virion diameter is about 60nm) or porcine epidemic diarrhea virus antigen (virion diameter is about 130nm).
  • the content of the present invention also includes the preparation method of the DC cell-based bifunctional nanobody, comprising the following steps:
  • the gene sequence of the porcine DC-specific nanobody was constructed by overlapping extension PCR method to construct a bifunctional nanobody expression system. After the expression product was purified, it was incubated with the pathogenic antigen to immunize pigs. After immunization, serum, lymph nodes and other samples were collected, and antibodies and cytokines were tested. Detection, for the evaluation of immune efficacy.
  • the content of the present invention also includes the bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells, and the bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells uses a linker element to encode a DC-specific nanobody After connecting the gene of the gene and the gene encoding the pig type O FMDV-specific Nanobody, the target gene fragment is further recombinantly expressed to obtain the target protein.
  • amino acid sequence of the porcine DC-specific nanobody Nb131 is shown in SEQ ID NO.:5
  • amino acid sequence of the porcine O-type FMDV-specific nanobody Nb104 is shown in SEQ ID NO.:6.
  • nucleotide sequence of the gene of the nanobody Nb131 specific for encoding pig DC is shown in SEQ ID NO.: 1, the nucleotide sequence of the gene for the nanobody Nb104 of the specificity of the pig DC type O FMDV Shown in SEQ ID NO.:2.
  • the linker element is a linker element (G4S) 4, the nucleotide sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 3, and the amino acid sequence of the linker element (G4S) 4 is as shown in SEQ ID NO. : As shown in 7.
  • amino acid sequence of the porcine O-type FMDV bifunctional nanobody Nb131-104 targeting porcine DC cells is shown in SEQ ID NO.:8.
  • the equilibrium dissociation constant (KD) of porcine DC-specific nanobody Nb131 and porcine DC cells is 3.43 ⁇ 10 -9
  • the equilibrium dissociation constant (KD) of porcine O-type FMDV antigen-specific nanobody Nb104 and porcine O-type FMDV antigen (KD) is 6.82 ⁇ 10 -10
  • the equilibrium dissociation constant (KD) of porcine DC/FMDV bifunctional nanobody Nb131-104 and porcine DC cells is 6.02 ⁇ 10 -8 respectively
  • the equilibrium dissociation constant (KD) between -104 and porcine O-type FMDV antigen is 2.41 ⁇ 10 -9 .
  • the content of the present invention also includes nucleic acid or gene, which encodes the porcine O-type FMDV bifunctional nanobody Nb131-104 targeting porcine DC cells, and its nucleotide sequence is shown in SEQ ID NO.:4.
  • the content of the present invention also includes the construction method of the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells.
  • the gene of antibody Nb131-104 was inserted into the pMECS vector, and then introduced into Escherichia coli WK6 competent cells to obtain recombinant bacteria; the recombinant bacteria were induced to express the target protein, and the recombinant bacteria were lysed and purified to obtain bifunctional nanobody Nb131-104.
  • the present invention uses the technical advantages of large library capacity and easy high-throughput screening by constructing a phage nanobody gene library to target the target protein of porcine CD205 , to screen porcine CD205-specific nanobodies, and carry out targeted presentation of antigens and evaluation of immune efficacy.
  • the present invention adopts a new technical idea, utilizes the specific combination between the antigen and the nanobody and the bridging effect of the bifunctional nanobody to realize the CD205 targeting of the antigen, and this method can avoid chemical coupling and gene fusion expression methods
  • the whole virus can be used to replace the conventional protein antigen, and the natural epitope of the whole virus can be fully utilized, which is easy to be applied in clinical practice.
  • the content of the present invention also includes the porcine O-type FMDV bifunctional nanobody targeting porcine CD205.
  • the porcine O-type FMDV bifunctional nanobody targeting porcine CD205 uses a linker element to encode the gene of the CD205 target protein-specific nanobody Nb193 and After the gene encoding the O-type FMDV antigen-specific nanobody Nb104 is connected, the target gene fragment is obtained by further recombinant expression to obtain the target protein.
  • nucleotide sequence of the pig CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.: 9
  • nucleotide sequence of the pig O-type FMDV antigen-specific nanobody Nb104 is shown in SEQ ID NO.: 2 shown.
  • amino acid sequence of the porcine CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.:11
  • the amino acid sequence of the porcine O-type FMDV antigen-specific nanobody Nb104 is shown in SEQ ID NO.:6.
  • the nucleotide sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 3
  • the amino acid sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 7.
  • the amino acid sequence of the porcine O-type FMDV bifunctional nanobody Nb193-104 targeting porcine CD205 is shown in SEQ ID NO.:12.
  • the equilibrium dissociation constant (K D ) of the porcine CD205-specific nanobody Nb193 and the porcine CD205 target protein is 1.04 ⁇ 10 -9
  • the equilibrium dissociation constant (K D ) of the porcine O-type FMDV-specific nanobody Nb104 and the porcine O-type FMDV antigen is The dissociation constant (K D ) was 6.82 ⁇ 10 -10 .
  • the content of the present invention also includes nucleic acid or gene, which encodes the porcine O-type FMDV bifunctional nanobody Nb193-104 targeting porcine CD205, the nucleotide sequence of which is shown in SEQ ID NO.:10.
  • the content of the present invention also includes the preparation method of the porcine O-type FMDV bifunctional nanobody targeting porcine CD205, comprising the following steps: inserting the coding gene of the bifunctional nanobody Nb193-104 into the pMECS vector, and then introducing it into Escherichia coli WK6 State cells were obtained to obtain recombinant bacteria; the recombinant bacteria were induced to express the target protein, and the recombinant bacteria were lysed and purified to obtain the bifunctional nanobody Nb193-104.
  • the content of the present invention also includes a porcine PEDV bifunctional nanobody targeting porcine CD205.
  • the bifunctional nanobody uses a linker element to connect the gene encoding the CD205 target protein-specific nanobody Nb193 and the coding porcine PEDV antigen-specific nanobody Nb2 Obtain the target protein obtained by further recombinant expression of the target gene fragment.
  • nucleotide sequence of the porcine CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.: 9
  • nucleotide sequence of the porcine PEDV antigen-specific nanobody Nb2 is shown in SEQ ID NO.: 13 shown.
  • amino acid sequence of the porcine CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.:11
  • the amino acid sequence of the porcine PEDV antigen-specific nanobody Nb2 is shown in SEQ ID NO.:14.
  • the linker element is a linker element (G4S) 4, the nucleotide sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 3, and the amino acid sequence of the linker element (G4S) 4 is as shown in SEQ ID NO. : As shown in 7.
  • the amino acid sequence of the porcine PEDV bifunctional nanobody Nb193-2 targeting porcine CD205 is shown in SEQ ID NO.:16.
  • the equilibrium dissociation constant (K D ) between the porcine CD205-specific nanobody Nb193 and the porcine CD205 target protein is 1.04 ⁇ 10 -9
  • the equilibrium dissociation constant (K D ) between the porcine PEDV-specific nanobody Nb2 and the porcine PEDV antigen ( K D ) is 1.03 ⁇ 10 -8
  • the equilibrium dissociation constant (K D ) of the porcine CD205/PEDV bifunctional nanobody Nb193-2 and the target protein of porcine CD205 is 1.52 ⁇ 10 -8
  • the porcine CD205 The equilibrium dissociation constant (K D ) of /PEDV bifunctional nanobody Nb193-2 and porcine PEDV antigen is 1.01 ⁇ 10 -8 .
  • the content of the present invention also includes nucleic acid or gene, which encodes the porcine PEDV bifunctional nanobody Nb193-2 targeting porcine CD205, the nucleotide sequence of which is shown in SEQ ID NO.:15.
  • the content of the present invention also includes the preparation method of the porcine PEDV bifunctional nanobody targeting porcine CD205, comprising the following steps: inserting the coding gene of the bifunctional nanobody Nb193-2 into the pMECS vector, and then introducing it into Escherichia coli WK6 competent cells , obtain the recombinant bacteria; induce the recombinant bacteria to express the target protein, lyse the recombinant bacteria and purify to obtain the bifunctional nanobody Nb193-2.
  • the contents of the present invention also include the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells, the porcine O-type FMDV bifunctional nanobody targeting porcine CD205, and the porcine CD205-targeting porcine bifunctional nanobody Application of the nucleic acid or gene described in the PEDV bifunctional nanobody in the preparation of a porcine vaccine.
  • the vectors of the present invention include but are not limited to pMECS vectors, other vectors, such as pHEN1, pHEN4, pComb3XSS, pCANTAB5e, pPIC9K, pYES2, etc., and viral vectors, such as baculovirus, lentiviral vector, adenoviral vector, AAV viral vector , Retrovirus, etc., as well as transposons and other gene transfer systems, according to the subsequent expression and mode of action, you can choose a variety of vector forms.
  • the cells of the present invention include but are not limited to Escherichia coli WK6 competent cells, other host cells such as Escherichia coli TOP10, BL21, XL1-blue in prokaryotic cells, eukaryotic cells CHO, 293, etc., and other hosts include brewing Yeast BY4743 cells, Pichia pastoris GS115 cells, and sf9 insect cells, etc.
  • the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells provided by the present invention is specifically realized through the following technical schemes:
  • a bifunctional nanobody expression system (Nb131-104) was constructed by overlapping extension PCR. After the expression product was purified, it was combined with porcine O-type Incubate with FMDV antigen, immunize pigs, collect samples such as serum and lymph nodes after immunization, and detect antibodies and cytokines for immune efficacy evaluation.
  • a kind of porcine O-type FMDV bifunctional nanobody targeting porcine CD205 provided by the invention is realized through the following technical solutions:
  • PCR technology was used to amplify the CysR-FNII truncated gene sequence of porcine CD205 molecule, and pET-32a expression vector was used to construct recombinant expression vector of porcine CD205 molecule, and the soluble expression and protein purification of prokaryotic system were carried out.
  • the purified porcine CD205 target protein was coated as an antigen, and phage display technology was used for 3-5 rounds of affinity screening to obtain a high-affinity porcine CD205-specific nanobody, named Nb193.
  • the porcine O-type FMDV antigen was coated, and 3-5 rounds of affinity screening were performed using phage display technology to obtain a high-affinity porcine O-type FMDV-specific nanobody, named Nb104.
  • pig CD205-specific nanobody Nb193 and pig O-type FMDV-specific nanobody Nb104 were used to construct a bifunctional nanobody expression system (Nb193-104) by overlapping extension PCR method. After the expression product was purified, it was mixed with pig O Incubate with FMDV antigen, immunize pigs, collect post-immunization serum, lymph nodes and other samples, and detect antibodies and cytokines for immune efficacy evaluation.
  • the porcine PEDV bifunctional nanobody targeting porcine CD205 provided by the present invention is realized according to the following technical scheme:
  • the porcine PEDV antigen was coated, and 3-5 rounds of affinity screening were performed using phage display technology to obtain a high-affinity porcine PEDV-specific nanobody, which was named Nb2.
  • the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells of the present invention can simultaneously specifically bind porcine DC cells and porcine O-type foot-and-mouth disease antigens, and is carried out in improving the immune efficacy of porcine O-type FMDV antigens Application can increase the titers of IgG and IgG1 antibodies after immunization of pig O-type FMDV antigen, prolong the duration of IgG antibodies after immunization, increase the proportion of CD4 + T cells and CD8 + T cells, and promote IFN- ⁇ , IL-2, IL- 4 secretion.
  • the porcine O-type FMDV bifunctional nanobody targeting porcine CD205 of the present invention uses a bifunctional nanobody that can specifically bind to porcine CD205 target protein and porcine O-type FMDV antigen at the same time to improve the immune efficacy of porcine O-type FMDV antigen
  • the application carried out can increase the IgG, IgG1, IgG2a antibody titer after immunization with pig O-type FMDV antigen, promote lymphocyte proliferation and IFN- ⁇ , IL-6, IL-4 secretion, and induce efficient cellular immune response.
  • porcine CD205-targeted porcine PEDV bifunctional nanobody of the present invention uses a bifunctional nanobody that can specifically bind to porcine CD205 target protein and porcine PEDV antigen at the same time to improve the immune efficacy of porcine PEDV antigen. It can improve the IgG, IgG1, IgG2a and mucosal IgA antibody titers after porcine PEDV antigen immunization, promote lymphocyte proliferation and IFN- ⁇ , IL-6, IL-4 secretion, and induce efficient cellular immune response.
  • the bifunctional nanobody of the present invention can be incubated and assembled directly with the antigen.
  • This method can not only use the whole virus to replace the protein antigen, make full use of the natural antigen epitope of the whole virus, but also avoid the chemical coupling and gene fusion expression methods.
  • the present invention also has strong versatility. By replacing different pathogen-specific nanobodies and then constructing corresponding porcine DC-targeting or CD205-targeting bifunctional nanobodies, it can be extended and applied to other pigs. with vaccine antigens.
  • the present invention has the following advantages: the bifunctional nanobody provided by the present invention, which can be used to target antigens to porcine DC cells or porcine CD205, is the first domestic and foreign bifunctional nanobody technology Means, using porcine DC cells or porcine CD205 molecules as the target to explore a new way to greatly improve the immune efficacy of porcine vaccine antigens, the invention helps to design a new, efficient, and actively targeted antigen delivery system for porcine DC cells.
  • the bifunctional nanobody is used as a technical link to directly incubate and assemble the antigen to realize DC or CD205 targeting of the antigen. This method can replace the traditional protein antigen with complete pathogenic particles, and fully utilize the natural properties of complete pathogenic particles.
  • Antigen epitopes can avoid the influence of conventional chemical coupling and gene fusion expression methods on the antigen structure.
  • the present invention has versatility, can screen specific nanobodies for different pathogens, and construct corresponding porcine DC-targeting bifunctional nanobodies, which can be extended and applied to other porcine vaccine antigens.
  • the operation is simple, efficient, and efficient.
  • the time-consuming is short, and the design and structure of the bifunctional nanobody are relatively clear, and it is easy to use the microbial genetic engineering system to efficiently produce, the manufacturing cost is low, and it has good stability.
  • the bifunctional nanobody and antigen can be used after incubation, which is simple and easy to implement, closer to veterinary clinical practice, and easy to popularize.
  • Figure 1 is the immunofluorescence image of porcine BMDC cells observed by laser confocal microscope. The cells were stained with PE anti-porcine CD11c, FITC anti-porcine CD11b and DAPI respectively, and the cell morphology was observed.
  • Fig. 2 is the result of PCR amplification of VHH gene.
  • M DL2000bp DNA marker
  • lanes 1 and 2 are amplification products of VHH gene fragments.
  • Fig. 3 is an electrophoresis diagram of identification of a single clone of a phage gene library.
  • lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, M: DL2000bp DNA marker.
  • Figure 4 is an indirect ELISA method for detecting the binding activity of Nanobodies.
  • the abscissa indicates different nanobody numbers, the ordinate indicates the OD450 value, the porcine DC fragmentation product represents the sample well, the porcine bone marrow progenitor cell fragmentation product represents the negative well, and Control represents the blank well.
  • Figure 5 is the SDS-PAGE electrophoresis and Western Blot identification results of the purified Nanobodies.
  • Lanes 1-6 in the left figure are the results of SDS-PAGE identification of randomly selected purified Nanobodies; M: protein standards; lanes 1-6 in the right figure are the results of Western Blot identification of randomly selected purified Nanobodies; M: protein standards.
  • Fig. 6 is the result of PCR amplification of VHH gene.
  • M DL2000bp DNA marker
  • lanes 1-5 are amplification products of VHH gene fragments.
  • Fig. 7 is an electrophoresis diagram of identification of a single clone of a phage gene library.
  • lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, M: DL2000bp DNA marker.
  • Figure 8 is an indirect ELISA method for detecting the binding activity of Nanobodies.
  • the abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, O-type FMDV indicates the sample well, BHK-21 indicates the negative well, and Control indicates the blank well.
  • Fig. 9 shows the specificity of detecting Nanobodies by indirect ELISA method.
  • the abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, type A FMDV and Asia1 type FMDV indicate the sample well, and Control indicates the blank well.
  • Figure 10 is the SDS-PAGE electrophoresis and Western Blot identification results of the purified Nanobodies.
  • Lanes 1-5 in the left figure are the results of SDS-PAGE identification of randomly selected purified Nanobodies; M: protein standards; lanes 1-5 in the right figure are the results of Western Blot identification of randomly selected purified Nanobodies; M: protein standards.
  • Figure 11 is the result of SOE-PCR amplification of the bifunctional Nanobody gene fragment.
  • M DL2000bp DNA marker
  • the other lane is the amplified product of bifunctional nanobody gene fragment.
  • Figure 12 is the SDS-PAGE electropherogram and Western Blot identification results of the purified bifunctional nanobody Nb131-104.
  • Lanes 1 and 2 in the figure above are the SDS-PAGE identification results of the purified bifunctional nanobody Nb131-104; lanes 1 and 2 in the figure below are the Western Blot identification results of the purified bifunctional nanobody Nb131-104; M :protein standards.
  • Figure 13 shows the binding ability of the bifunctional nanobody Nb131-104 to porcine BMDC observed by confocal laser microscopy.
  • the upper 4 groups of pictures are the results of confocal laser microscope observation of the test group added with bifunctional nanobodies, and the lower 4 groups of pictures are the results of laser confocal microscope observation of the blank control group.
  • the cells were stained with AF647 anti-porcine CD1, FITC Nb131-104 and DAPI, respectively, and the cell morphology was observed.
  • Fig. 14 shows the FMDV antigen delivery ability of bifunctional nanobody Nb131-104 observed by laser confocal microscope.
  • the upper 4 groups of pictures are the results of confocal laser microscope observation of the test group added with bifunctional nanobodies, and the lower 4 groups of pictures are the results of laser confocal microscope observation of the blank control group.
  • the cells were stained with AF647 anti-porcine CD1, FITC Nb131-104 and DAPI, respectively, and the cell morphology was observed.
  • Figure 15 shows the level of specific antibodies in serum after immunization detected by ELISA, and blood was collected on days 14, 28, 42, and 56 after immunization for antibody detection.
  • Figure 16 is the ELISA detection of IgG1 antibody levels in serum after immunization, and blood samples were collected for antibody detection on 14 days, 28 days, 42 days, and 56 days after immunization.
  • Figure 17 ELISA to detect the level of IgG2a antibody in the serum after immunization, and blood samples were collected on days 14, 28, 42, and 56 after immunization for antibody detection.
  • Figure 18 ELISA to detect the duration of antibodies after immunization, and blood was collected on days 14, 28, 42, and 56 after immunization for antibody detection.
  • Figure 20 PCR amplification results of CysR-FNII truncated gene of porcine CD205 molecule.
  • M is DL2000 DNA marker
  • lane 1 is the amplified product of the CysR-FNII truncated gene fragment of porcine CD205 molecule.
  • FIG. 21 SDS-PAGE electrophoresis and Western Blot identification results of the purified porcine CD205 target protein.
  • Lane 1 is the purified porcine CD205 target protein, and M is protein standards.
  • Fig. 22 PCR amplification results of VHH gene.
  • M is DL2000 DNA marker
  • lanes 1 and 2 are amplification products of VHH gene fragments.
  • Fig. 23 Electropherogram of identification of single clone of phage gene library.
  • lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, and M is DL2000 DNA marker.
  • Figure 24 Indirect ELISA method to detect the binding activity of Nanobodies.
  • the abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, the porcine CD205 target protein indicates the sample well, any other irrelevant protein prepared under the same conditions indicates the negative well, and Control indicates the blank well.
  • FIG. 25 SDS-PAGE electrophoresis and Western Blot identification results of purified Nanobodies. Lanes 1 and 2 are randomly selected purified nanobodies, and M is protein standards.
  • Fig. 26 PCR amplification results of VHH gene.
  • M is DL2000 DNA marker
  • lanes 1-3 are amplification products of VHH gene fragments.
  • Fig. 27 Electropherogram of identification of single clone of phage gene library.
  • lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, and M is DL2000 DNA marker.
  • Fig. 28 Indirect ELISA method to detect the binding activity of Nanobodies.
  • the abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, PEDV indicates the sample well, ST indicates the negative well, and Control indicates the blank well.
  • Figure 29 Indirect ELISA method to detect the specificity of Nanobodies.
  • the abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, O-FMDV, PCV2, PRRSV, PRV indicate the sample well, and Control indicates the blank well.
  • FIG. 30 SDS-PAGE electrophoresis and Western Blot identification results of purified Nanobodies. Lanes 1 and 2 are randomly selected purified nanobodies, and M is protein standards.
  • Fig. 31 SOE-PCR amplification results of bifunctional nanobody gene fragments.
  • M is DL10000 DNA marker
  • lane 1 is the amplified product of bifunctional nanobody gene fragment.
  • FIG. 32 SDS-PAGE electrophoresis and Western Blot identification results of the purified bifunctional nanobody Nb193-2.
  • Lane 1 is the purified bifunctional nanobody Nb193-2, and M is protein standards.
  • Figure 33 Immunofluorescence of porcine BMDC cells observed by laser confocal microscope. The cells were stained with PE anti-porcine CD11c, FITC anti-porcine CD11b and DAPI, respectively, and the cell morphology was observed.
  • Fig. 34 Observation of the binding ability of bifunctional nanobody 193-2 to porcine BMDC by laser confocal microscope.
  • the upper 4 groups of pictures are the results of confocal laser microscope observation of the test group added with bifunctional nanobodies, and the lower 4 groups of pictures are the results of laser confocal microscope observation of the blank control group.
  • the cells were stained with AF647 anti-porcine CD1, FITC Nb193-2 and DAPI, respectively, and the cell morphology was observed.
  • Figure 36 ELISA detection of IgG1 antibody level in serum 28 days after immunization.
  • Figure 37 ELISA detection of IgG2a antibody level in serum 28 days after immunization.
  • Figure 38 ELISA detection of mucosal IgA antibody levels 28 days after immunization.
  • Figure 39 MTT method to detect the level of lymphocyte proliferation.
  • Figure 40 ELISA detection of IFN- ⁇ , IL-6, IL-4 secretion levels in the cell culture supernatant.
  • porcine bone marrow progenitor cells (4 ⁇ 10 6 cells/mL), use porcine recombinant GM-CSF (20ng/mL, purchased from Shanghai Unionwell Company) and porcine recombinant IL-4 (10ng/mL, purchased from Shanghai Unionville Company) to induce differentiation, and after continuous culture for 7 days, porcine bone marrow-derived dendritic cells (BMDC) were collected and planted in cell culture dishes (2 ⁇ 10 6 cells/mL), and treated with 4% paraformaldehyde Fix at room temperature for 10 min, wash with PBS three times, place in PBS containing 0.1% Triton for 5 min at 37°C, wash with PBS for three times, place in blocking solution for 1 h at 37°C, and use PE-labeled anti-porcine CD11c fluorescent antibody (1 Incubate at 4°C for 30 min at 4°C, wash cells three times with PBS, and incubate for 30 min at 4°C with FITC-labeled anti-porcine CD11
  • the cells were washed three times with PBS, stained with DAPI (working solution, purchased from Shanghai Biyuntian Company) for 10 min, washed with PBS three times and placed under a confocal laser microscope for observation, as shown in Figure 1, a relatively abundant Pig BMDC cells.
  • DAPI working solution, purchased from Shanghai Biyuntian Company
  • porcine BMDC cells After repeated induction and differentiation for many times, a sufficient amount of porcine BMDC cells (about 1 ⁇ 10 10 cells) were ultrasonically disrupted, centrifuged at 12,000 g for 10 min, discarded the supernatant, and resuspended the pellet with normal saline to obtain porcine BMDC cell disruption products.
  • porcine BMDC cell crushing product 1 mg was mixed with Freund's complete adjuvant in equal volume to immunize a Xinjiang Bactrian camel; 1 week later, 1 mg of porcine BMDC cell crushing product was mixed with Freund's incomplete adjuvant in equal volume to immunize the camel Bactrian camels were immunized once a week for a total of 6 times to stimulate the body to produce specific antibodies against porcine BMDC cells; after the immunization, 100mL camel peripheral blood lymphocytes were extracted to extract the total RNA of lymphocytes, followed by the reverse transcription kit (purchased from TAKARA Company) described the operation and synthesized cDNA.
  • the VHH fragment was amplified by PCR.
  • the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, and 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered.
  • VHH and V HHR as upstream and downstream primers to amplify the VHH fragment.
  • the reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C.
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 2, a VHH gene fragment of about 350 bp was seen, which was consistent with the expected size.
  • the target band was purified and recovered using a gel recovery kit (purchased from TAKARA).
  • VHH gene fragment was digested with Pst I and Not I, and then ligated into pMECS vector (purchased from Novagen).
  • the ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers.
  • Example 2 Take 200 ⁇ L of the phage display library prepared in Example 1 that was frozen at -80°C and inoculate it into 500 mL of 2 ⁇ TY medium. After culturing for 3-5 hours at 37°C with a shaker speed of 200 rpm, add 50 ⁇ L of helper phage VCSM13 (purchased from Novagen), incubated at 37° C. for 1 h, and then cultured overnight at 37° C. with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified phage display library. The amplified phage display library was resuspended in 5mL 0.1M PBS buffer to obtain its suspension.
  • helper phage VCSM13 purchased from Novagen
  • the bound phage was eluted, and infected with 5 times the volume (about 500 ⁇ L) of Escherichia coli TG1 cells in the logarithmic growth phase (OD 600 of 0.8), cultured at 37°C for 1 h, and added 50 ⁇ L of helper phage VCSM13 (purchased from Novagen) to invade TG1 cells were transfected, and the supernatant was obtained by centrifugation to obtain the phages selected in the first round, which were used for the next round of screening. A total of 3 rounds of the same screening process were carried out. 10 ⁇ L of the phages obtained in each round of screening were applied to LB solid medium and cultured overnight at 37°C for observing the enrichment process of affinity screening. As shown in Table 2, after three rounds of affinity screening of the library, more phages were enriched in each round of screening than in the previous round.
  • Affinity screening rounds Amount of phage library input (pfu/mL) Amount of recovered phage library (pfu/mL) First round of affinity screening 1.12 ⁇ 10 7 8.71 ⁇ 10 3 Second round of affinity screening 1.04 ⁇ 10 7 6.25 ⁇ 10 4 The third round of affinity screening 1.16 ⁇ 10 7 3.04 ⁇ 10 5
  • the bacteria were lysed by ultrasonication, and the lysate was taken after centrifugation to obtain the nanobodies expressed by the recombinant bacteria against the crushed products of porcine BMDC cells.
  • the numbers of each nanobody were 1-200.
  • the indirect ELISA reaction was used to identify the binding activity of each Nanobody numbered 1-200 to the broken product of porcine BMDC cells.
  • the porcine BMDC cell disruption product that 10 ⁇ g embodiment 1 prepares joins 10mL concentration and is the NaHCO of 100mM in the solution (pH8.2), mixes homogeneously, gets 100 ⁇ L and joins in each sample hole of 96-well microtiter plate, in 4 °C for overnight coating, and in the control wells, the broken product of porcine bone marrow progenitor cells was used to replace the broken product of porcine BMDC cells for coating; the next day, discard the liquid in the plate, wash 5 times with PBS buffer containing 0.05% Tween-20, and shoot Dry, add 100 ⁇ L of 5% skim milk solution to each well, block at room temperature for 2 h; wash 5 times with PBS buffer containing 0.05% Tween-20, add 100 ⁇ L of each nanobody to each well of the ELISA plate in turn, incuba
  • the recombinant bacterial plasmids expressing positive Nanobodies against porcine BMDCs obtained in Example 3 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42° C. Cultivate under conditions for 1 hour, centrifuge and concentrate the bacterial solution, spread it on an LB plate containing 100 ⁇ g/mL ampicillin, and incubate at 37°C for 12 to 16 hours; pick a single colony, and obtain recombinants expressing nanobodies targeting porcine BMDC Bacteria A1-A17.
  • Nanobodies (numbered 18, 29, 37, 47, 60, 64) were randomly selected for SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 5 that the nanobodies have obvious bands at about 16kD, consistent with the expected size of the target fragment, and the purity is over 90%.
  • the affinities of the obtained 17 nanobodies against porcine BMDCs and the disrupted products of porcine BMDCs were identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare).
  • a Biacore TM X100 protein interaction instrument purchased from GE Healthcare.
  • the purified nanobodies (numbered as shown in Table 3) were serially diluted from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and respectively combined with the fragmented products coupled to the CM5 chip
  • the binding time is 180s, and ethanolamine is used for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethane
  • the detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 3.
  • the nanobody numbered 131 has a K D value of 3.43 ⁇ 10 -9 , which is the nanobody with the highest affinity for porcine BMDC obtained through the screening of the present invention. Subsequent studies will be carried out around this nanobody.
  • ka represents the association rate constant
  • kd represents the dissociation rate constant
  • K D represents the equilibrium dissociation constant
  • the recombinant bacterial plasmid of nanobody Nb131 was extracted and sent to Shanghai Shenggong Company for sequence determination to obtain its nucleotide sequence and amino acid sequence.
  • the nucleotide sequence and amino acid sequence are respectively shown as SEQ ID NO.:1 and SEQ ID NO. :5 shown.
  • the VHH fragment was amplified by PCR.
  • the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, and 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered.
  • VHH gene fragment with a size of about 750bp as a template, and use V HHF and V HHR as upstream and downstream primers to amplify the VHH fragment.
  • the reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C.
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 6, a VHH gene fragment of about 350 bp was seen, which was consistent with the expected size. Purify and recover the target bands by using the instruction manual of the gel recovery kit (purchased from TAKARA company).
  • VHH gene fragment was digested with PstI and NotI, and then ligated into pMESC vector (purchased from Novagen).
  • the ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers.
  • the phage was eluted, and infected with 5 times the volume (about 500 ⁇ L) of Escherichia coli TG1 cells in logarithmic growth phase (OD 600 is 0.8), cultured at 37°C for 1 hour, and added 50 ⁇ L of helper phage VCSM13 (purchased from Novagen) to infect TG1
  • the cells were centrifuged to take the supernatant, and the phages selected in the first round were obtained for the next round of screening. A total of 3 rounds of the same screening process were carried out. 10 ⁇ L of the phages obtained in each round of screening were applied to LB solid medium and cultured overnight at 37°C for observing the enrichment process of affinity screening. As shown in Table 4, after three rounds of affinity screening of the library, more phages were enriched in each round of screening than in the previous round.
  • Affinity screening rounds Amount of phage library input (pfu/mL) Amount of recovered phage library (pfu/mL) First round of affinity screening 1.01 ⁇ 10 7 3.96 ⁇ 10 3 Second round of affinity screening 1.12 ⁇ 10 7 2.58 ⁇ 10 4 The third round of affinity screening 1.07 ⁇ 10 7 1.49 ⁇ 10 5
  • the indirect ELISA reaction was used to identify the binding activity of each nanobody to type O foot-and-mouth disease virus.
  • Add 10 ⁇ g of O-type foot-and-mouth disease inactivated virus to 10 mL of 100 mM NaHCO 3 solution (pH8.2), mix well, take 100 ⁇ L and add it to each well of a 96-well microtiter plate, and coat overnight at 4 ° C to set up BHK- 21 cell disruption products were used as a control; the next day, the liquid in the plate was discarded, washed 5 times with PBS buffer containing 0.05% Tween-20, patted dry, and 100 ⁇ L of 5% skim milk solution was added to each well, and blocked at room temperature for 2 hours; Wash 5 times with PBS buffer containing 0.05% Tween-20, add each Nanobody to each well of the ELISA plate in turn, incubate at room temperature for 1 hour, wash off unbound Nanobodies with PBS buffer containing 0.05% Tween-20 100 ⁇ L
  • the cross-reactivity between each nanobody and type A and Asia1 foot-and-mouth disease virus was detected respectively, and the corresponding OD 450 was measured by a microplate reader.
  • the difference is that the ELISA plate For the coating antigen, type A and Asia1 foot-and-mouth disease virus were used instead of type O foot-and-mouth disease virus.
  • the results are shown in Figure 9, among the nanobodies obtained in the present invention, there are 8 nanobodies that have cross-reactivity to type A foot-and-mouth disease virus, 9 nanobodies have cross-reactivity to Asia1 type foot-and-mouth disease virus, and 3 nanobodies have cross-reactivity to type A foot-and-mouth disease virus.
  • Type A and Asia1 type foot-and-mouth disease viruses all have cross-reactivity, and all the other 15 nanobodies have extremely low cross-reactivity to type A and Asia1 type foot-and-mouth disease viruses, indicating that the present invention has obtained specific nanobodies for O-type foot-and-mouth disease viruses, and at the same time, Some nanobodies had cross-reactivity with type A and Asia1 foot-and-mouth disease virus.
  • the specific nanobody recombinant bacterial plasmids for O-type foot-and-mouth disease virus obtained in Example 7 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42 ° C, respectively, at 37 ° C and the shaker speed was 200 rpm Cultivate for 1 hour under the condition of 1 h, centrifuge and concentrate the bacterial solution, spread it on the LB plate containing 100 ⁇ g/mL ampicillin, and culture at 37 ° C for 12 to 16 hours; pick a single colony, and obtain 15 expression targets for O-type foot-and-mouth disease virus Nanobody recombinant bacteria B1-B15.
  • the bacterial cell precipitate was collected by centrifugation, and the nanobody crude extract was obtained by ultrasonic crushing, and the nanobody was purified by affinity chromatography using a nickel column (purchased from GE Healthcare).
  • Purified Nanobodies (numbered 26, 43, 45, 82, 88) were randomly selected for SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 10 that the nanobodies have obvious bands at about 16kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
  • the affinities of the obtained 15 nanobodies to the inactivated type O foot-and-mouth disease virus were identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare).
  • a Biacore TM X100 protein interaction instrument purchased from GE Healthcare.
  • the detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 5.
  • the Nanobody No. 104 has a K D value of 6.82 ⁇ 10 -10 , which is the Nanobody with the highest affinity obtained through the screening of the present invention. Subsequent studies will be carried out around this Nanobody.
  • ka represents the association rate constant
  • kd represents the dissociation rate constant
  • K D represents the equilibrium dissociation constant
  • the recombinant bacterial plasmid of nanobody Nb104 was extracted and sent to Shanghai Shenggong Company for sequence determination to obtain its gene sequence and amino acid sequence.
  • the gene sequence and amino acid sequence are shown in SEQ ID NO.:2 and SEQ ID NO.:6 respectively .
  • the recombinant bacterial plasmids expressing porcine DC-specific nanobody Nb131 and porcine O-type foot-and-mouth disease virus-specific nanobody Nb104 were extracted respectively, and amplified using the primers NbF, NbLR, NbLF, and NbR shown in Table 6 to obtain porcine DC
  • the gene fragments of the specific nanobody Nb131 and the porcine O-type foot-and-mouth disease virus specific nanobody Nb104 were purified and recovered.
  • the bifunctional nanobody fragment Nb131-104 was constructed by splicing by Overlap Extension (SOE) PCR method, and the reaction was divided into two steps conduct:
  • the first step reaction is carried out without primers, and the reaction conditions are: 95°C for 3min; 95°C for 30s, 62°C for 30s, 72°C for 2min, a total of 8 cycles; 72°C for 10min;
  • the second step of the reaction only needs to add primers NbF and NbR to the original PCR tube.
  • the reaction conditions are: 95°C for 3min; 95°C for 30s, 55°C for 30s, 72°C for 2min, a total of 25 cycles; 72°C for 10min;
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light.
  • a gene fragment of about 900bp was seen, which was consistent with the expected fragment size, and then inserted into the pMECS vector (purchased from Novagen) between the Pst I and Xba I restriction sites, transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C, and cultured for 1h at 37°C with a shaker speed of 200rpm , concentrate the bacterial solution by centrifugation, spread it on an LB plate containing 100 ⁇ g/mL ampicillin, and incubate at 37° C. for 12 to 16 hours; select a single colony to obtain recombinant bacteria C expressing bifunctional nanobodies.
  • the specific sequence of each primer is shown in Table 6:
  • the plasmid expressing the recombinant bacteria C of the bifunctional nanobody Nb131-104 was extracted and sent to Shanghai Sangong Company for sequence determination to obtain the nucleotide sequence and amino acid sequence of the linker element (G4S) 4 and the bifunctional nanobody Nb131-104.
  • the nucleotide sequences of the linker element (G4S) 4 and the bifunctional nanobody Nb131-104 are shown in SEQ ID NO.:3 and SEQ ID NO.:4 respectively, and the linker element (G4S) 4 and the bifunctional nanobody Nb131-
  • the amino acid sequences of 104 are shown in SEQ ID NO.:7 and SEQ ID NO.:8 respectively.
  • the bifunctional nanobody Nb131-104 was prepared by recombinant bacteria.
  • Biacore TM X100 protein interaction instrument (purchased from GE Healthcare) was used to identify the affinity of the bifunctional nanobody Nb131-104 to the broken product of porcine BMDC cells and type O foot-and-mouth disease virus, respectively.
  • 1 mg of porcine BMDC cell breakdown product and 1 mg of O-type foot-and-mouth disease virus were coupled to the CM5 chip using the coupling reagent N-ethyl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma).
  • the purified bifunctional nanobodies were serially diluted from 100nM in normal saline (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM respectively), and combined with the fragmented products coupled to the chip respectively, the binding time 180s, use ethanolamine to block, utilize HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v /v) Surfactant P20) was washed to remove unbound material at a flow rate of 30 ⁇ L/min and regenerated with 10 mM glycine/HCl (pH 2.5).
  • HBS-EP buffer 10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM ED
  • the detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 7.
  • the equilibrium dissociation constants (K D ) of bifunctional nanobody Nb131-104 with porcine BMDC cell breakdown product and type O foot-and-mouth disease virus were 6.02 ⁇ 10 -8 and 2.41 ⁇ 10 -9 , respectively.
  • ka represents the association rate constant
  • kd represents the dissociation rate constant
  • K D represents the equilibrium dissociation constant
  • the bifunctional nanobody Nb131-104 prepared in Example 9 was subjected to column chromatography to remove endotoxin, and an endotoxin detection kit (purchased from Pyrosate, 0.25EU/mL) was used to detect the endotoxin content to ensure that the endotoxin level was less than 0.05 EU, and labeled with FITC fluorescent dye (purchased from Shanghai Univ Company), and ultrafiltered (4000g, 20min) to remove unlabeled excess dye.
  • an endotoxin detection kit purchased from Pyrosate, 0.25EU/mL
  • FITC fluorescent dye purchased from Shanghai Univ Company
  • the O-type FMDV inactivated antigen was purified by sucrose density gradient centrifugation, and the purified product was identified by SDS-PAGE and Western Blotting.
  • Fresh porcine BMDC cells were induced to differentiate according to the method described in Example 1, and the bifunctional nanobody Nb131-104 (5 ⁇ g/mL) labeled with FITC fluorescent dye (purchased from Shanghai Youningwei Company) and the purified FMDV inactivated antigen were used (5 ⁇ g/mL) at 4°C for 30min, then incubated with porcine BMDC cells (1 ⁇ 10 6 cells/mL) at 4°C for 30min, collected cells, fixed with 4% paraformaldehyde at room temperature for 10min, washed three times with PBS, and placed in Treat in PBS containing 0.1% Triton at 37°C for 5 min, wash with PBS three times, place in blocking solution for 1 h at 37°C, wash cells with PBS three times, use AF647-labeled anti-pig CD1
  • the pig After incubating the bifunctional nanobody Nb131-104 (5 ⁇ g/mL) prepared in Example 9 with the purified FMDV inactivated antigen (5 ⁇ g/mL) at 4° C. for 60 min, supplemented with 206 immune adjuvant, the pig was immunized. The neck muscle of each pig was immunized with 2mL, and blood samples were collected at different time points (14d, 28d, 42d, 56d).
  • the bifunctional nanobody Nb131-104 was compatible with the PBS control group, and the non-DC targeting nanobody was compatible with FMDV Control group, non-DC targeting nanobody compatibility PBS control group, FMDV control group and PBS blank control group, 5 pigs in each group.
  • FMD liquid-phase blocking ELISA antibody detection technology has good sensitivity, rapid diagnosis and reproducibility, and has a good correlation with challenge protection. It has been widely used in the detection of FMD immune antibody levels.
  • FAO and OIE recommends the use of liquid-phase blocking ELISA method to evaluate the immune effect of FMD, so this study uses liquid-phase blocking ELISA method to evaluate the immune effect of FMD.
  • the FMD liquid-phase blocking ELISA antibody detection kit was used to detect the specific antibody level, antibody subtype (IgG1 and IgG2a) and the duration of immunity in the serum after immunization, and the detection steps were all operated according to the kit instructions.
  • the test results are shown in Figure 15.
  • the antibody titers of the bifunctional nanobody Nb131-104 compatible with FMDV test group were significantly higher than those of other test groups and control groups after immunization, indicating that the bifunctional nanobody can promote the production of antibodies to FMDV antigen after immunization.
  • Antibody subtype detection results show (as shown in Figure 16, 17), the IgG1 antibody titer of the bifunctional nanobody Nb131-104 compatibility FMDV test group after immunization is all significantly higher than other test groups and control groups, and the IgG2a antibody titer There was no significant difference between the valency and other FMDV test groups, indicating that the bifunctional nanobody Nb131-104 can significantly increase the IgG1 antibody level after immunization with FMDV antigen, and the effect on increasing the IgG2a antibody level was not significantly different from other test groups; the results of the duration of immunity test showed that (As shown in Figure 18), the antibody level of the bifunctional nanobody Nb131-104 compatibility FMDV test group can last up to 56 days after immunization, and the antibody titers are significantly higher than other test groups and control groups, indicating that the bifunctional nanobody Nb131-104 can not only significantly increase the antibody level of FMDV antigen after immunization, but also maintain a high level of
  • lymph nodes of immunized pigs were collected near the injection site, and lymph node single cell suspension was prepared. Lymphocyte separation medium was added to the cell suspension, and density gradient centrifugation (3000g, 20min) was carried out. Divide into four layers, absorb the second layer), wash 3 times with serum-containing cell culture medium and inoculate into 96-well plate (1 ⁇ 10 6 cells/mL); separate lymphocytes with anti-CD4 Stained with CD8 fluorescent antibody, the proliferation results were detected by FACS Aria flow cytometer, and the data were acquired by CellQuest software.
  • the secretion levels of IFN- ⁇ , IL-2, and IL-4 in the Nb131-104+O-FMDV test group were significantly higher than those of the rest of the test group and the control group, further illustrating that both The functional nanobody Nb131-104 can significantly improve the cellular immune response induced by FMDV antigen immunization.
  • porcine spleen tissue under aseptic conditions extract the total RNA of spleen tissue, operate according to the instructions of the reverse transcription kit (purchased from TAKARA company), synthesize cDNA, and use the primers F1 and R1 designed in Title 1 of this example to perform PCR Amplify to obtain a gene fragment with a size of about 600bp.
  • the PCR reaction conditions were: 95°C for 3min; 95°C for 30s, 59°C for 1min, 72°C for 1min, a total of 30 cycles; 72°C for 10min.
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 20, it can be seen that the CysR-FNII truncated gene fragment of the porcine CD205 molecule of about 600bp is in line with the expected Same size.
  • the target band was purified and recovered using a gel recovery kit (purchased from TAKARA).
  • the purified and recovered CysR-FNII gene fragment of porcine CD205 molecule was digested with BamHI and HindIII, and then ligated into pET-32a vector (purchased from Novagen).
  • the ligation product was transformed into E.coli BL21 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial solution was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, and cultured at 37°C for 12 ⁇ 16 hours; pick a single colony to obtain recombinant bacterium 1 expressing the target protein of porcine CD205.
  • the target protein of porcine CD205 was prepared by recombinant strain 1.
  • the recombinant bacterium 1 was crushed, and the lysate of the bacterium was taken as the crude extract of porcine CD205 target protein, and the target porcine CD205 protein was purified by affinity chromatography using a nickel column (purchased from GE Healthcare).
  • the purified protein was identified by SDS-PAGE electrophoresis and Western blot. It can be seen from Figure 21 that the target protein of porcine CD205 has an obvious band at about 23kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
  • porcine CD205 target protein prepared in Example 13 and mix it with complete Freund's adjuvant in equal volumes to immunize a Xinjiang Bactrian camel; after 1 week, mix 1 mg of porcine CD205 target protein with Freund's incomplete adjuvant The volume was mixed, and the Bactrian camel was immunized once a week for a total of 6 times to stimulate the body to produce specific antibodies against the target protein of porcine CD205; cDNA was synthesized according to the instructions of the reverse transcription kit (purchased from TAKARA).
  • the VHH fragment was amplified by PCR.
  • the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered.
  • VHH and V HHR as upstream and downstream primers to amplify the VHH fragment.
  • the reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C.
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 22, a VHH gene fragment of about 350 bp was seen, which was consistent with the expected size.
  • the target band was purified and recovered using a gel recovery kit (purchased from TAKARA).
  • VHH gene fragment was digested with Pst I and Not I, and then ligated into pMECS vector (purchased from Novagen).
  • the ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers. The results are shown in Figure 23.
  • Example 14 Take 200 ⁇ L of the phage display library prepared in Example 14 that was frozen at -80°C and inoculate it into 500 mL of 2 ⁇ TY medium. After culturing for 3-5 hours at 37°C with a shaker speed of 200 rpm, add 50 ⁇ L of helper phage VCSM13 (purchased from Novagen), incubated at 37° C. for 1 h, and then cultured overnight at 37° C. with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified phage display library. The amplified phage display library was resuspended in 5mL 0.1M PBS buffer to obtain its suspension.
  • helper phage VCSM13 purchased from Novagen
  • Affinity screening rounds Amount of phage library input (pfu/mL) Amount of recovered phage library (pfu/mL) First round of affinity screening 1.1 ⁇ 10 7 5.3 ⁇ 10 3 Second round of affinity screening 1.4 ⁇ 10 7 2.2 ⁇ 10 4 The third round of affinity screening 1.2 ⁇ 10 7 1.6 ⁇ 10 5
  • the indirect ELISA reaction was used to identify the binding activity of the nanobodies numbered 1-200 to the target protein of porcine CD205.
  • the plasmids of recombinant bacteria expressing nanobodies obtained in Example 16 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C, and cultured at 37°C and a shaker speed of 200rpm for 1h. Concentrate the bacterial solution by centrifugation, spread it on an LB plate containing 100 ⁇ g/mL ampicillin, and incubate at 37°C for 12-16 hours; pick a single colony, and obtain recombinant bacteria A1-A14 expressing nanobodies.
  • the purified nanobodies were subjected to SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 25 that the nanobodies have obvious bands at about 16kD, consistent with the expected size of the target fragment, and the purity is over 90%.
  • the affinity of each nanobody to the porcine CD205 target protein was identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare).
  • a Biacore TM X100 protein interaction instrument purchased from GE Healthcare.
  • the purified Nanobodies (numbered as shown in Table 4) were serially diluted from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and respectively mixed with the fragmented products coupled to the chip
  • the binding time is 180s, and ethanolamine is used for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-
  • the test results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 12.
  • the Nanobody No. 193 has a K D value of 1.04 ⁇ 10 -9 , which is the Nanobody with the highest affinity obtained through the screening of the present invention. Subsequent studies will be carried out around this Nanobody.
  • ka represents the association rate constant
  • kd represents the dissociation rate constant
  • K D represents the equilibrium dissociation constant
  • the recombinant bacterial plasmid of Nanobody 193 was extracted and sent to Shanghai Shenggong Company for sequence determination to obtain its gene sequence and amino acid sequence.
  • the gene sequence and amino acid sequence are shown in SEQ ID NO.:9 and SEQ ID NO.:11 respectively .
  • the VHH fragment was amplified by PCR.
  • the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, and 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered.
  • VHH and V HHR as upstream and downstream primers to amplify the VHH fragment.
  • the reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C.
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 26, a VHH gene fragment of about 350bp was seen, which was consistent with the expected size. Purify and recover the target bands by using the instruction manual of the gel recovery kit (purchased from TAKARA company).
  • VHH gene fragment was digested with Pst I and Not I, and then ligated into pMESC vector (purchased from Novagen).
  • the ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers.
  • Example 18 Take 200 ⁇ L of the phage library prepared in Example 18 that was frozen at -80°C and inoculate it into 500 mL of 2 ⁇ TY medium. After culturing for 3-5 hours at 37°C with a shaker speed of 200 rpm, add 50 ⁇ L of helper phage VCSM13 ( (purchased from Novagen), incubated at 37°C for 1 h, and then cultured overnight at 37°C with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified phage library. Resuspend the amplified phage library in 5mL 0.1M PBS to obtain its suspension.
  • helper phage VCSM13 (purchased from Novagen)
  • PEG6000 purchased from Shanghai Sangon Co., Ltd.
  • Affinity screening rounds Amount of phage library input (pfu/mL) Amount of recovered phage library (pfu/mL) First round of affinity screening 1.2 ⁇ 10 7 4.1 ⁇ 10 3 Second round of affinity screening 1.1 ⁇ 10 7 1.8 ⁇ 10 4 The third round of affinity screening 1.1 ⁇ 10 7 2.4 ⁇ 10 5
  • each nanobody to porcine PEDV antigen was identified by indirect ELISA reaction.
  • the OD 450 value of the sample well is more than 2.5 times greater than the OD 450 value of the control well, it is judged as a positive clone well.
  • the results are shown in Figure 28.
  • a total of 11 Nanobodies can specifically bind to the porcine PEDV antigen (in order to screen for high-affinity Nanobodies, only Nanobodies with an OD 450 value greater than 2.0 were selected).
  • the cross-reactivity between each nanobody and O-type FMDV, PCV2, PRRSV, and PRV was detected respectively, and the corresponding OD 450 was measured by a microplate reader.
  • the difference is that the ELISA
  • the coating antigen of the plate was replaced by PEDV with O-type FMDV, PCV2, PRRSV and PRV.
  • the results are shown in Figure 29, the nanobodies obtained by the present invention have extremely low cross-reactivity to O-type FMDV, PCV2, PRRSV, and PRV, indicating that the present invention has obtained specific nanobodies against porcine PEDV.
  • the plasmids of the specific nanobody recombinant bacteria directed against porcine PEDV antigen obtained in Example 20 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C respectively, at 37°C and the shaker speed was 200rpm Cultivate for 1 hour under the condition of 1 h, centrifuge and concentrate the bacterial liquid, spread it on LB plates containing 100 ⁇ g/mL ampicillin, and incubate at 37°C for 12 to 16 hours; select a single colony to obtain recombinant bacteria expressing nanobodies B1- B11.
  • Nanobodies were subjected to SDS-PAGE electrophoresis and Western blot identification. From Figure 30, it can be seen that the Nanobodies have obvious bands at about 16kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
  • the test results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 14.
  • the Nanobody No. 2 has a K D value of 1.03 ⁇ 10 -8 , which is the Nanobody with the highest affinity obtained through the screening of the present invention. Subsequent studies will be carried out around this Nanobody.
  • ka represents the association rate constant
  • kd represents the dissociation rate constant
  • K D represents the equilibrium dissociation constant
  • the plasmids of recombinant bacteria expressing porcine CD205-specific nanobody 193 and porcine PEDV antigen-specific nanobody 2 were extracted respectively, and amplified using the primers NbF, NbLR, NbLF, and NbR shown in Table 15 to obtain porcine CD205-specific Nanobody 193 and porcine PEDV antigen-specific nanobody 2 gene fragments were purified and recovered.
  • the bifunctional nanobody fragment Nb193-2 was constructed by splicing by Overlap Extension (SOE) PCR method, and the reaction was divided into two steps conduct:
  • the first step reaction is carried out without primers, and the reaction conditions are: 95°C for 3min; 95°C for 30s, 62°C for 30s, 72°C for 2min, a total of 8 cycles; 72°C for 10min;
  • the second step of the reaction only needs to add primers NbF and NbR to the original PCR tube.
  • the reaction conditions are: 95°C for 3min; 95°C for 30s, 55°C for 30s, 72°C for 2min, a total of 25 cycles; 72°C for 10min;
  • the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light.
  • a gene fragment of about 900bp was seen, which was consistent with the expected fragment size, and then inserted into the pMECS vector (purchased from Novagen) between the Pst I and Xba I restriction sites, transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C, and cultured for 1h at 37°C with a shaker speed of 200rpm , concentrate the bacterial solution by centrifugation, spread it on an LB plate containing 100 ⁇ g/mL ampicillin, and incubate at 37° C. for 12 to 16 hours; select a single colony to obtain recombinant bacteria C expressing bifunctional nanobodies.
  • the specific sequence of each primer is shown in Table 15:
  • the plasmid expressing the recombinant bacteria C of the bifunctional nanobody Nb193-2 was extracted and sent to Shanghai Sangong Company for sequence determination to obtain the gene sequence and amino acid sequence of the linker element (G4S) 4 and the bifunctional nanobody Nb193-2.
  • the gene sequences of linker element (G4S) 4 and bifunctional Nanobody Nb193-2 are shown in SEQ ID NO.: 3 and SEQ ID NO.: 15 respectively, and the gene sequences of linker element (G4S) 4 and bifunctional Nanobody Nb193-2
  • the amino acid sequences are respectively shown in SEQ ID NO.:7 and SEQ ID NO.:16.
  • the bifunctional nanobody Nb193-2 was prepared by recombinant bacteria.
  • the affinities of the bifunctional nanobody 193-2 to the porcine CD205 target protein and the porcine PEDV antigen were respectively identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare).
  • a Biacore TM X100 protein interaction instrument purchased from GE Healthcare.
  • the coupling reagent N-eth yl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide purchased from Sigma
  • physiological Saline diluted the purified bifunctional nanobody from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and combined with the broken products coupled to the chip respectively
  • the binding time was 180s , using ethanolamine for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150m
  • the detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 8.
  • the equilibrium dissociation constants (K D ) of bifunctional nanobody Nb193-2 with porcine CD205 target protein and porcine PEDV antigen are 1.52 ⁇ 10 -8 and 1.01 ⁇ 10 -8 , respectively.
  • ka represents the association rate constant
  • kd represents the dissociation rate constant
  • K D represents the equilibrium dissociation constant
  • porcine BMDC cells Collect fresh porcine bone marrow progenitor cells (4 ⁇ 10 6 cells/mL), use porcine recombinant GM-CSF (20ng/mL, purchased from Shanghai Unionwell Company) and porcine recombinant IL-4 (10ng/mL, purchased from Shanghai Unionville Company) to induce differentiation, and after continuous culture for 7 days, porcine BMDC cells were collected and planted in cell culture dishes (2 ⁇ 10 6 cells/mL), fixed with 4% paraformaldehyde at room temperature for 10 min, and washed with PBS for 3 After one pass, they were treated in PBS containing 0.1% Triton at 37°C for 5 minutes, washed three times with PBS, and blocked in blocking solution for 1 hour at 37°C, and PE-labeled anti-porcine CD11c fluorescent antibody (diluted at 1:1000, purchased from Shanghai Unionville Company) was incubated at 4°C for 30 min, cells were washed three times with PBS, and FITC-labeled anti-porcine CD
  • the bifunctional nanobody Nb193-2 prepared in Example 22 was subjected to column chromatography to remove endotoxin, and the endotoxin content was detected using an endotoxin detection kit (purchased from Pyrosate Company, 0.25EU/mL) to ensure the endotoxin level It is less than 0.05 EU, and it is labeled with FITC fluorescent dye (purchased from Shanghai Unionwell Co., Ltd.), and the unlabeled excess dye is removed by ultrafiltration (4000g, 20min).
  • the porcine PEDV antigen was purified by sucrose density gradient centrifugation, and the purified product was identified by SDS-PAGE and Western Blotting.
  • porcine BMDC cells were induced to differentiate, and FITC fluorescent dye-labeled bifunctional nanobody (5 ⁇ g/mL) was used to incubate with purified PEDV antigen (5 ⁇ g/mL) for 30 minutes at 4° C., and then Incubate with porcine BMDC cells (1 ⁇ 10 6 cells/mL) at 4°C for 30 min, collect the cells, fix with 4% paraformaldehyde at room temperature for 10 min, wash with PBS three times, and place in PBS containing 0.1% Triton for 5 min at 37°C.
  • porcine BMDC cells (1 ⁇ 10 6 cells/mL) at 4°C for 30 min, collect the cells, fix with 4% paraformaldehyde at room temperature for 10 min, wash with PBS three times, and place in PBS containing 0.1% Triton for 5 min at 37°C.
  • the pig After incubating the bifunctional nanobody Nb193-2 (5 ⁇ g/mL) prepared in Example 22 and the purified PEDV antigen (5 ⁇ g/mL) at 4°C for 60 min, supplemented with 206 immune adjuvant, the pig was immunized with an experiment. Pig neck muscles were immunized with 2 mL, and blood samples were collected on the 28th day.
  • a bifunctional nanobody-compatible PBS control group, a non-CD205-targeting nanobody-compatible PEDV control group, a non-CD205-targeting nanobody-compatible PBS control group, and a PEDV control group were set. group and PBS blank control group, with 5 pigs in each group.
  • the PEDV antibody detection kit (purchased from Wuhan Keqian Company) was used to detect the specific antibody level, antibody subtype (IgG1 and IgG2a) and IgA antibody level in the serum after immunization, and the detection steps were all performed according to the kit instructions. The results of antibody detection are shown in Figure 35.
  • the antibody titer of the bifunctional nanobody-compatible PEDV test group 28 days after immunization was significantly higher than that of the other test groups and the control group, indicating that the bifunctional nanobody can promote the production of antibodies to PEDV antigens after immunization ;
  • Antibody subtype detection results showed (as shown in Figure 36, 37), the IgG1 and IgG2a antibody titers of the bifunctional nanobody compatibility PEDV test group were significantly higher than other test groups and control groups 28 days after immunization, indicating that the bifunctional nanobody Nanobodies can significantly increase the levels of IgG1 and IgG2a antibodies after PEDV antigen immunization;
  • IgA test results show (as shown in Figure 38), although the antibody levels of each test group and control group are low, the bifunctional nanobody compatibility PEDV test group
  • the titer of mucosal IgA antibody 28 days after immunization was significantly higher than that of other test groups and control groups, indicating that the bifunctional nano
  • lymph nodes of immunized pigs were collected near the injection site, and lymph node single cell suspension was prepared. Lymphocyte separation medium was added to the cell suspension, and density gradient centrifugation (3000g, 20min) was carried out. Divided into four layers in total, aspirate the second layer), washed 3 times with serum-containing cell culture medium and inoculated into 96-well plates (1 ⁇ 10 6 cells/mL); purified PEDV antigen (5 ⁇ g /mL), LPS stimulated cells, placed in a cell culture incubator (37°C, containing 5% CO 2 ) and continued to culture for 48h; MTT (5mg/mL) was added to each well and cultured for 4h, cell samples were collected by centrifugation, and DMSO was added And mix until the crystals are dissolved, and place the cell plate in a microplate reader to read the results. The results are shown in Figure 39. Compared with the control group, the Nb193-2+PEDV test group can significantly increase the proliferation level of lymphocytes and induce

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Abstract

Disclosed in the present invention are a bifunctional nanobody based on DC, and a construction method therefor and the use thereof. The bifunctional nanobody is a target protein, wherein a target protein specific nanobody based on DC is linked to a gene encoding a virus antigen specific nanobody with a particle size of 150 nm or less using a linker element to obtain a target gene fragment, and the obtained target protein is subjected to recombinant expression. According to the method of the present invention, specific nanobodies for different pathogens are screened, and a corresponding porcine DC targeted bifunctional nanobody is constructed and is used for preparing a vaccine for pigs.

Description

基于DC细胞的双功能纳米抗体及其构建方法和应用DC cell-based bifunctional nanobody and its construction method and application 技术领域technical field
本发明属于生物技术领域，具体涉及基于DC细胞的双功能纳米抗体及其构建方法和应用。The invention belongs to the field of biotechnology, and in particular relates to a DC cell-based bifunctional nanobody and its construction method and application.
背景技术Background technique
树突状细胞(dendritic cell，DC)是机体内功能最强大的专职抗原提呈细胞，针对DC细胞实现抗原的主动靶向输送对于提高抗原提呈效率和改善抗原免疫效力具有重要意义，是新型疫苗制剂研制与应用领域的前沿热点。抗原的DC靶向主要是通过靶向DC细胞表面受体来实现的，外源性抗原经特异性抗体或配体靶向至DC细胞后，DC细胞对其进行摄取、加工处理及提呈，循MHCⅡ或交叉提呈途径，与MHC分子组装形成肽段-MHC复合物，运送至细胞表面后，分别提呈至相应的效应细胞，激活免疫反应。与非靶向抗原相比较，靶向抗原在提高抗原提呈效率以及减少抗原用量方面都具有显著改善，甚至可以循交叉提呈途径，诱导CD8 +T细胞免疫应答，在抗病毒感染和肿瘤治疗方面具有良好的应用价值，目前多个DC靶向人用疫苗已进入临床试验阶段。 Dendritic cells (DC) are the most powerful professional antigen-presenting cells in the body. The active targeted delivery of antigens to DC cells is of great significance for improving the efficiency of antigen presentation and improving the efficacy of antigen immunity. Frontier hotspots in the field of development and application of new vaccine preparations. The DC targeting of antigens is mainly achieved by targeting DC cell surface receptors. After exogenous antigens are targeted to DC cells by specific antibodies or ligands, DC cells absorb, process and present them. Following the MHC II or cross-presentation pathway, it is assembled with MHC molecules to form a peptide-MHC complex, which is transported to the cell surface and presented to the corresponding effector cells to activate the immune response. Compared with non-targeted antigens, targeted antigens have significantly improved antigen presentation efficiency and reduced antigen dosage, and can even induce CD8 + T cell immune responses through cross-presentation pathways, which are useful in antiviral infection and tumor treatment. It has good application value, and several DC-targeted human vaccines have entered the clinical trial stage.
CD205作为DC特异性的抗原提呈受体，属于C型凝集素受体(C-type lectin receptors，CLRs)中的巨噬细胞甘露糖受体家族，又称为DEC-205或Ly75，其在组织中分布广泛，高度表达于DC细胞和胸腺上皮细胞，在B细胞、T细胞、NK细胞和巨噬细胞中很少表达或几乎不表达，CD205是目前在机体淋巴器官T细胞区域的DC细胞中表达最广泛的唯一受体，在抗原提呈过程中发挥着重要作用。As a DC-specific antigen-presenting receptor, CD205 belongs to the macrophage mannose receptor family in C-type lectin receptors (CLRs), also known as DEC-205 or Ly75, which is in Widely distributed in tissues, highly expressed in DC cells and thymus epithelial cells, rarely expressed or hardly expressed in B cells, T cells, NK cells and macrophages, CD205 is currently the DC cell in the T cell region of the body's lymphoid organs The most widely expressed unique receptor in , which plays an important role in antigen presentation.
研究显示，与非靶向抗原相比较，CD205靶向可以使OVA抗原提呈效率提高至少100倍，抗原用量减少近1000倍；通过将乙肝病毒preS抗原靶向至CD205，可以在小鼠体内产生高效的IgG1和IgG2a抗体反应，对乙肝病毒感染产生预防和治疗效果；分别将登革热病毒NS1抗原和EDⅢ抗原靶向至CD205，可以在小鼠体内激发高效的抗体反应和T细胞反应，产生较好的攻毒保护作用；而在人类黑色素瘤临床试验中发现，通过将NY-ESO-1抗原靶向至CD205，可以产生高效的体液免疫反应和细胞免疫反应，部分患者病症表现稳定，并出现肿瘤衰退以及病灶缩小。综上所述，CD205靶向可以减少抗原用量，诱导高效的免疫应答，在抗病毒感染和肿瘤治疗方面具有良好的发展潜力，CD205已成为新型疫苗制剂研发与应用领域的重要靶点，CD205靶向人用疫苗也已进入临床试验阶段。Studies have shown that compared with non-targeted antigens, CD205 targeting can increase the presentation efficiency of OVA antigens by at least 100 times, and reduce the amount of antigens by nearly 1000 times; by targeting HBV preS antigens to CD205, it can produce Efficient IgG1 and IgG2a antibody responses have preventive and therapeutic effects on hepatitis B virus infection; targeting dengue virus NS1 antigen and EDⅢ antigen to CD205 can stimulate efficient antibody responses and T cell responses in mice, resulting in better In human melanoma clinical trials, it was found that by targeting NY-ESO-1 antigen to CD205, highly efficient humoral and cellular immune responses could be generated, and some patients showed stable symptoms and tumors Regression and shrinkage of lesions. In summary, CD205 targeting can reduce the amount of antigen used, induce efficient immune response, and has good development potential in antiviral infection and tumor therapy. CD205 has become an important target in the development and application of new vaccine preparations. CD205 targeting The vaccine for human use has also entered the stage of clinical trials.
目前，关于猪DC靶向研究、猪DC细胞表面受体种类、结构和功能研究较少。现有研究报道中由于靶向抗原的不同，产生的免疫效果也不尽相同，且靶向提呈作用机制尚不清楚，靶向抗原的设计也存在不足，不能产生较为理想的免疫效果。作为DC特异性的抗原提呈受体，CD205靶向在兽医领域研究日益受到关注。通过将立克次氏体MSP1a抗原靶向至牛CD205，可以显著提高IFN-γ水平和抗体水平；通过将禽流感病毒HA抗原靶向至鸡CD205，在免后14天可以产生高效的抗体水平；通过将裂谷热病毒eGn抗原靶向至羊CD205，尽管可以提高IFN-γ水平，但降低了抗体水平以及攻毒保护作用；通过将PRRSV的GP345M融合抗原靶向至猪CD205，尽管可以产生较好的IFN-γ水平和抗体水平，但不能产生攻毒保护作用，猪肺部病变差异不显著；CD205靶向FMDV、PEDV研究均未见报道。综上所述，由于物种以及靶向抗原的不同，产生的免疫效果也不尽相同；而关于猪CD205靶向研究极少，作用机制尚不清楚，且靶向抗原的设计也存在不足，现有研究报道中多使用蛋白质抗原作为靶向抗原，蛋白质抗原由于抗原表位有限，往往不能产生较为理想的免疫效果。因此，如何设计更加有效的靶向抗 原对于改善DC靶向免疫效力将发挥重要作用。抗原的DC靶向主要是利用DC表面受体相应的特异性单克隆抗体或单链抗体，通过化学偶联或基因融合表达的方法与蛋白质抗原进行组装，该过程较为繁琐，易影响抗原结构。因此，探索新的抗原组装策略对于改善DC靶向免疫效力具有重要意义。At present, there are few studies on porcine DC targeting, porcine DC cell surface receptor types, structure and function. In the existing research reports, due to the different target antigens, the immune effects are not the same, and the mechanism of target presentation is still unclear, and the design of the target antigen is also insufficient, which cannot produce a more ideal immune effect. As a DC-specific antigen-presenting receptor, CD205 targeting has attracted increasing attention in the field of veterinary medicine. By targeting Rickettsia MSP1a antigen to bovine CD205, IFN-γ levels and antibody levels can be significantly increased; by targeting avian influenza virus HA antigen to chicken CD205, highly effective antibody levels can be produced 14 days after immunization ;By targeting the Rift Valley fever virus eGn antigen to sheep CD205, although IFN-γ levels can be increased, antibody levels and challenge protection are reduced; by targeting the GP345M fusion antigen of PRRSV to pig CD205, although it can produce Better IFN-γ level and antibody level, but can not produce challenge protection, and the difference in lung lesions of pigs is not significant; CD205 targeting FMDV, PEDV studies have not been reported. To sum up, due to the differences in species and target antigens, the immune effects are not the same; however, there are very few studies on porcine CD205 targeting, the mechanism of action is still unclear, and the design of target antigens is also insufficient. In some research reports, protein antigens are often used as target antigens. Due to the limited epitopes of protein antigens, protein antigens often cannot produce ideal immune effects. Therefore, how to design more effective targeting antigens will play an important role in improving the efficacy of DC-targeted immunity. The DC targeting of antigens mainly uses specific monoclonal antibodies or single-chain antibodies corresponding to DC surface receptors to assemble with protein antigens through chemical coupling or gene fusion expression. This process is cumbersome and easily affects the antigen structure. Therefore, exploring new antigen assembly strategies is of great significance for improving the efficacy of DC-targeted immunity.
纳米抗体作为目前已知的具有完整功能的最小抗原结合片段，与单克隆抗体和单链抗体相比，具有相对分子质量小(约15KDa)、免疫原性低、稳定性高、易于利用微生物基因工程系统高效生产等特点，在研制廉价高效的治疗性抗体和检测试剂方面具有良好的应用前景，通过基因工程改造而来的多价和多特异性纳米抗体解决了单价纳米抗体血清稳定性差、体内半衰期短等不足，同时又可以利用不同的靶点协同促进免疫反应，被许多权威研究机构认为是治疗癌症的潜在应用药物。纳米抗体在DC靶向方面的研究表现出了较好的应用前景，研究表明，通过制备针对MHCⅡ、CD11b和CD36的特异性纳米抗体，分别利用GFP、酵母泛素、OVA和流感病毒HA进行DC靶向研究，均可以产生较好的免疫反应，在小鼠体内可以产生针对流感病毒的攻毒保护作用；通过制备小鼠CD206特异性纳米抗体，可以实现纳米凝胶和报告基因的靶向提呈。截止到目前为止，在猪DC或CD205靶向研究方面，仍然以单克隆抗体和单链抗体为主，猪DC或CD205特异性纳米抗体未见报道。Nanobodies are the smallest antigen-binding fragments known to have complete functions. Compared with monoclonal antibodies and single-chain antibodies, nanobodies have a relatively small molecular weight (about 15KDa), low immunogenicity, high stability, and easy utilization of microbial genes. With the characteristics of high-efficiency production of engineering systems, it has good application prospects in the development of cheap and efficient therapeutic antibodies and detection reagents. The multivalent and multispecific nanobodies transformed by genetic engineering solve the problem of poor serum stability of monovalent nanobodies and problems in vivo. Short half-life and other shortcomings, and at the same time, different targets can be used to synergistically promote the immune response, which is considered by many authoritative research institutions as a potential drug for the treatment of cancer. The study of nanobodies in DC targeting has shown promising application prospects. Studies have shown that by preparing specific nanobodies against MHC Ⅱ, CD11b, and CD36, GFP, yeast ubiquitin, OVA, and influenza virus HA were used to target DCs, respectively. Targeted research can produce a better immune response, and can produce a protective effect against influenza virus in mice; by preparing mouse CD206-specific nanobodies, the targeted extraction of nanogels and reporter genes can be achieved. presented. So far, in the research of targeting porcine DC or CD205, monoclonal antibodies and single-chain antibodies are still the mainstays, and porcine DC or CD205-specific nanobodies have not been reported.
目前，关于猪DC细胞特异性纳米抗体及猪DC细胞靶向型双功能纳米抗体均未见报道，关于猪CD205特异性纳米抗体及猪CD205靶向双功能纳米抗体也均未见报道。At present, there are no reports about porcine DC cell-specific nanobodies and porcine DC cell-targeting bifunctional nanobodies, nor about porcine CD205-specific nanobodies and porcine CD205-targeting bifunctional nanobodies.
发明内容Contents of the invention
发明目的：本发明所要解决的技术问题是提供了可用于抗原靶向猪DC细胞的双功能纳米抗体。本发明采用一种新的技术思路，利用抗原与纳米抗体之间的特异性结合以及双功能纳米抗体的桥连作用实现抗原的DC靶向，该方法既可以避免化学偶联和基因融合表达方法对抗原结构的影响，又可以采用完整的病原粒子替代常规的蛋白质抗原，充分利用完整的病原粒子的天然抗原表位，易于在临床实际中开展应用。Purpose of the invention: The technical problem to be solved by the present invention is to provide a bifunctional nanobody that can be used for antigen-targeting porcine DC cells. The present invention adopts a new technical idea, utilizes the specific combination between the antigen and the nanobody and the bridging effect of the bifunctional nanobody to realize the DC targeting of the antigen, and this method can avoid chemical coupling and gene fusion expression methods For the impact on the antigen structure, the complete pathogenic particle can be used to replace the conventional protein antigen, and the natural epitope of the complete pathogenic particle can be fully utilized, which is easy to be applied in clinical practice.
本发明还要解决的技术问题是提供了所述的双功能纳米抗体的构建方法。The technical problem to be solved in the present invention is to provide a method for constructing the bifunctional nanobody.
本发明最后要解决的技术问题是提供了所述的双功能纳米抗体的应用。The final technical problem to be solved by the present invention is to provide the application of the bifunctional nanobody.
技术方案：为解决现有技术中存在的技术问题，本发明提供了基于DC细胞的双功能纳米抗体，所述双功能纳米抗体是以接头元件将基于DC细胞的目的蛋白特异性纳米抗体的基因和编码粒径为150nm以下的病毒抗原特异性纳米抗体的基因连接后获得目的基因片段并重组表达获得的目的蛋白。Technical solution: In order to solve the technical problems existing in the prior art, the present invention provides a DC cell-based bifunctional nanobody. The bifunctional nanobody uses a linker element to transfer the gene of a DC cell-based target protein-specific nanobody to The target gene fragment is obtained after being connected with the gene encoding the virus antigen-specific nanobody with a particle size below 150nm, and the obtained target protein is recombinantly expressed.
其中，所述基于DC细胞的目的蛋白特异性纳米抗体包括编码DC细胞特异性的纳米抗体或编码DC细胞抗原提呈受体特异性的纳米抗体。Wherein, the DC cell-based target protein-specific Nanobody includes a Nanobody encoding DC cell specificity or a Nanobody encoding DC cell antigen-presenting receptor specificity.
其中，所述猪DC细胞的目的蛋白特异性纳米抗体和粒径在150nm以内的猪病毒抗原特异性纳米抗体是利用噬菌体展示技术分别筛选获得。Wherein, the target protein-specific nanobody of the porcine DC cells and the porcine virus antigen-specific nanobody with a particle size within 150 nm are screened separately by using phage display technology.
进一步地，所述猪病毒抗原粒径为17nm～130nm。Further, the particle size of the porcine virus antigen is 17nm-130nm.
其中，所述猪病毒抗原包括猪圆环病毒抗原(病毒粒子直径约为17nm)、猪细小病毒抗原(病毒粒子直径约为20nm)、猪口蹄疫病毒抗原(病毒粒子直径约为25nm)、猪瘟病毒抗原(病毒粒子直径约为50nm)、猪繁殖与呼吸综合征病毒抗原(病毒粒子直径约为60nm)或猪流行性腹泻病毒抗原(病毒粒子直径约为130nm)中的一种或几种。Wherein, said porcine virus antigen comprises porcine circovirus antigen (diameter of virus particle is about 17nm), porcine parvovirus antigen (diameter of virus particle is about 20nm), porcine foot-and-mouth disease virus antigen (diameter of virus particle is about 25nm), classical swine fever One or more of viral antigens (virion diameter is about 50nm), porcine reproductive and respiratory syndrome virus antigen (virion diameter is about 60nm) or porcine epidemic diarrhea virus antigen (virion diameter is about 130nm).
本发明内容还包括所述的基于DC细胞的双功能纳米抗体的制备方法，包括以下步骤：The content of the present invention also includes the preparation method of the DC cell-based bifunctional nanobody, comprising the following steps:
1)使用新鲜诱导分化的DC细胞免疫骆驼或羊驼，经过多次免疫后，利用分子生物学原理提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌 体展示纳米抗体基因文库；1) Use freshly differentiated DC cells to immunize camels or alpacas. After multiple immunizations, use the principles of molecular biology to extract the cDNA of peripheral blood lymphocytes, amplify the antibody heavy chain variable region gene library, and construct phage display Nanobody gene library;
2)将新鲜诱导分化的DC细胞作为抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个猪DC特异性纳米抗体；2) Coat freshly induced differentiated DC cells as antigens, and use phage display technology to perform 3-5 rounds of affinity screening to obtain a porcine DC-specific nanobody;
3)使用所述的病毒免疫骆驼或羊驼，经过多次免疫后，利用分子生物学原理提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库；3) Use the virus to immunize camels or alpacas, and after multiple immunizations, use the principles of molecular biology to extract the cDNA of peripheral blood lymphocytes, and amplify the antibody heavy chain variable region gene library to construct phage display nanobodies gene library;
4)将所述的病毒作为抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个所述的病毒特异性纳米抗体；4) Coating the virus as an antigen, and using phage display technology to perform 3-5 rounds of affinity screening to obtain one of the virus-specific nanobodies;
5)将猪DC特异性纳米抗体的基因序列和所述的病毒特异性纳米抗体的基因序列连接后获得目的基因片段并重组表达获得的目的蛋白：利用猪DC特异性纳米抗体和该病原特异性纳米抗体的基因序列经重叠延伸PCR方法构建双功能纳米抗体表达体系，表达产物经纯化后，与该病原抗原孵育，对猪进行免疫，采集免后血清、淋巴结等样品，对抗体、细胞因子进行检测，用于免疫效力评价。5) After linking the gene sequence of the porcine DC-specific nanobody and the gene sequence of the virus-specific nanobody, obtain the target gene fragment and recombinantly express the obtained target protein: using the porcine DC-specific nanobody and the pathogen-specific The gene sequence of the nanobody was constructed by overlapping extension PCR method to construct a bifunctional nanobody expression system. After the expression product was purified, it was incubated with the pathogenic antigen to immunize pigs. After immunization, serum, lymph nodes and other samples were collected, and antibodies and cytokines were tested. Detection, for the evaluation of immune efficacy.
本发明内容还包括靶向猪DC细胞的猪O型FMDV的双功能纳米抗体，所述靶向猪DC细胞的猪O型FMDV的双功能纳米抗体是以接头元件将编码DC特异性的纳米抗体的基因和编码猪O型FMDV特异性的纳米抗体的基因连接后获得目的基因片段进一步重组表达获得的目的蛋白。The content of the present invention also includes the bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells, and the bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells uses a linker element to encode a DC-specific nanobody After connecting the gene of the gene and the gene encoding the pig type O FMDV-specific Nanobody, the target gene fragment is further recombinantly expressed to obtain the target protein.
其中，所述猪DC特异性的纳米抗体Nb131的氨基酸序列为SEQ ID NO.:5所示，所述猪O型FMDV特异性的纳米抗体Nb104的氨基酸序列为SEQ ID NO.:6所示。Wherein, the amino acid sequence of the porcine DC-specific nanobody Nb131 is shown in SEQ ID NO.:5, and the amino acid sequence of the porcine O-type FMDV-specific nanobody Nb104 is shown in SEQ ID NO.:6.
其中，所述编码猪DC特异性的纳米抗体Nb131的基因的核苷酸序列为SEQ ID NO.:1所示，所述编码猪O型FMDV特异性的纳米抗体Nb104的基因的核苷酸序列为SEQ ID NO.:2所示。Wherein, the nucleotide sequence of the gene of the nanobody Nb131 specific for encoding pig DC is shown in SEQ ID NO.: 1, the nucleotide sequence of the gene for the nanobody Nb104 of the specificity of the pig DC type O FMDV Shown in SEQ ID NO.:2.
其中，所述接头元件包括但不仅限于接头元件(G4S)4，还可以包括常见的(G4S)n，n＝1～6，还有驼源抗体IgG2c的铰链区、人抗体IgA的铰链区等，所述接头元件为接头元件(G4S)4，所述接头元件(G4S)4的核苷酸序列如SEQ ID NO.:3所示，接头元件(G4S)4的氨基酸序列如SEQ ID NO.:7所示。Wherein, the linker element includes but not limited to linker element (G4S)4, and may also include common (G4S)n, n=1-6, as well as the hinge region of camel-derived antibody IgG2c, the hinge region of human antibody IgA, etc. , the linker element is a linker element (G4S) 4, the nucleotide sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 3, and the amino acid sequence of the linker element (G4S) 4 is as shown in SEQ ID NO. : As shown in 7.
其中，所述靶向猪DC细胞的猪O型FMDV双功能纳米抗体Nb131-104的氨基酸序列如SEQ ID NO.:8所示。Wherein, the amino acid sequence of the porcine O-type FMDV bifunctional nanobody Nb131-104 targeting porcine DC cells is shown in SEQ ID NO.:8.
其中，猪DC特异性的纳米抗体Nb131与猪DC细胞的平衡解离常数(KD)为3.43×10 -9，猪O型FMDV抗原特异性纳米抗体Nb104与猪O型FMDV抗原的平衡解离常数(KD)为6.82×10 -10，猪DC/FMDV双功能纳米抗体Nb131-104与猪DC细胞的平衡解离常数(KD)分别为6.02×10 -8，猪DC/FMDV双功能纳米抗体Nb131-104与猪O型FMDV抗原的平衡解离常数(KD)为2.41×10 -9。 Among them, the equilibrium dissociation constant (KD) of porcine DC-specific nanobody Nb131 and porcine DC cells is 3.43×10 -9 , and the equilibrium dissociation constant (KD) of porcine O-type FMDV antigen-specific nanobody Nb104 and porcine O-type FMDV antigen (KD) is 6.82×10 -10 , the equilibrium dissociation constant (KD) of porcine DC/FMDV bifunctional nanobody Nb131-104 and porcine DC cells is 6.02×10 -8 respectively, and porcine DC/FMDV bifunctional nanobody Nb131 The equilibrium dissociation constant (KD) between -104 and porcine O-type FMDV antigen is 2.41×10 -9 .
本发明内容还包括核酸或基因，其编码所述的靶向猪DC细胞的猪O型FMDV双功能纳米抗体Nb131-104，其核苷酸序列如SEQ ID NO.:4所示。The content of the present invention also includes nucleic acid or gene, which encodes the porcine O-type FMDV bifunctional nanobody Nb131-104 targeting porcine DC cells, and its nucleotide sequence is shown in SEQ ID NO.:4.
本发明内容还包括所述的靶向猪DC细胞的猪O型FMDV双功能纳米抗体的构建方法，所述构建方法包括如下步骤：将编码靶向猪DC细胞的猪O型FMDV的双功能纳米抗体Nb131-104的基因插入pMECS载体，然后导入大肠杆菌WK6感受态细胞，获得重组菌；诱导重组菌表达目的蛋白，裂解重组菌后纯化获得双功能纳米抗体Nb131-104。The content of the present invention also includes the construction method of the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells. The gene of antibody Nb131-104 was inserted into the pMECS vector, and then introduced into Escherichia coli WK6 competent cells to obtain recombinant bacteria; the recombinant bacteria were induced to express the target protein, and the recombinant bacteria were lysed and purified to obtain bifunctional nanobody Nb131-104.
为了通过抗原的猪CD205靶向提呈进而改善抗原的免疫效力，本发明通过构建噬菌体纳米抗体基因文库，运用其库容量大、易高通量筛选的技术优势，以猪CD205目的蛋白为靶点，筛选猪CD205特异性纳米抗体，开展抗原的靶向提呈与免疫效力评价。 本发明采用一种新的技术思路，利用抗原与纳米抗体之间的特异性结合以及双功能纳米抗体的桥连作用实现抗原的CD205靶向，该方法既可以避免化学偶联和基因融合表达方法对抗原结构的影响，又可以采用全病毒替代常规的蛋白质抗原，充分利用全病毒的天然抗原表位，易于在临床实际中开展应用。In order to improve the immune efficacy of the antigen through the porcine CD205 targeted presentation of the antigen, the present invention uses the technical advantages of large library capacity and easy high-throughput screening by constructing a phage nanobody gene library to target the target protein of porcine CD205 , to screen porcine CD205-specific nanobodies, and carry out targeted presentation of antigens and evaluation of immune efficacy. The present invention adopts a new technical idea, utilizes the specific combination between the antigen and the nanobody and the bridging effect of the bifunctional nanobody to realize the CD205 targeting of the antigen, and this method can avoid chemical coupling and gene fusion expression methods For the impact on the antigen structure, the whole virus can be used to replace the conventional protein antigen, and the natural epitope of the whole virus can be fully utilized, which is easy to be applied in clinical practice.
本发明内容还包括靶向猪CD205的猪O型FMDV双功能纳米抗体，所述靶向猪CD205的猪O型FMDV双功能纳米抗体以接头元件将编码CD205目的蛋白特异性纳米抗体Nb193的基因和编码O型FMDV抗原特异性纳米抗体Nb104的基因连接后获得目的基因片段进一步重组表达获得的目的蛋白。The content of the present invention also includes the porcine O-type FMDV bifunctional nanobody targeting porcine CD205. The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 uses a linker element to encode the gene of the CD205 target protein-specific nanobody Nb193 and After the gene encoding the O-type FMDV antigen-specific nanobody Nb104 is connected, the target gene fragment is obtained by further recombinant expression to obtain the target protein.
其中，所述猪CD205目的蛋白特异性纳米抗体Nb193的核苷酸序列如SEQ ID NO.:9所示，猪O型FMDV抗原特异性纳米抗体Nb104的核苷酸序列如SEQ ID NO.:2所示。Wherein, the nucleotide sequence of the pig CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.: 9, and the nucleotide sequence of the pig O-type FMDV antigen-specific nanobody Nb104 is shown in SEQ ID NO.: 2 shown.
其中，所述猪CD205目的蛋白特异性纳米抗体Nb193的氨基酸序列如SEQ ID NO.:11所示，猪O型FMDV抗原特异性纳米抗体Nb104的氨基酸序列如SEQ ID NO.:6所示。Wherein, the amino acid sequence of the porcine CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.:11, and the amino acid sequence of the porcine O-type FMDV antigen-specific nanobody Nb104 is shown in SEQ ID NO.:6.
其中，所述接头元件包括但不仅限于接头元件(G4S)4，还可以包括常见的(G4S)n，n＝1～6，还有驼源抗体IgG2c的铰链区、人抗体IgA的铰链区等，所述接头元件(G4S)4的核苷酸序列如SEQ ID NO.:3所示，接头元件(G4S)4的氨基酸序列如SEQ ID NO.:7所示。Wherein, the linker element includes but not limited to linker element (G4S)4, and may also include common (G4S)n, n=1-6, as well as the hinge region of camel-derived antibody IgG2c, the hinge region of human antibody IgA, etc. , the nucleotide sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 3, and the amino acid sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 7.
其中，所述靶向猪CD205的猪O型FMDV双功能纳米抗体Nb193-104的氨基酸序列如SEQ ID NO.:12所示。其中，所述的猪CD205特异性纳米抗体Nb193与猪CD205目的蛋白的平衡解离常数(K D)为1.04×10 -9，猪O型FMDV特异性纳米抗体Nb104与猪O型FMDV抗原的平衡解离常数(K D)为6.82×10 -10。 Wherein, the amino acid sequence of the porcine O-type FMDV bifunctional nanobody Nb193-104 targeting porcine CD205 is shown in SEQ ID NO.:12. Wherein, the equilibrium dissociation constant (K D ) of the porcine CD205-specific nanobody Nb193 and the porcine CD205 target protein is 1.04×10 -9 , and the equilibrium dissociation constant (K D ) of the porcine O-type FMDV-specific nanobody Nb104 and the porcine O-type FMDV antigen is The dissociation constant (K D ) was 6.82×10 -10 .
本发明内容还包括核酸或基因，其编码所述的靶向猪CD205的猪O型FMDV双功能纳米抗体Nb193-104，其核苷酸序列如SEQ ID NO.:10所示。The content of the present invention also includes nucleic acid or gene, which encodes the porcine O-type FMDV bifunctional nanobody Nb193-104 targeting porcine CD205, the nucleotide sequence of which is shown in SEQ ID NO.:10.
本发明内容还包括所述的靶向猪CD205的猪O型FMDV双功能纳米抗体的制备方法，包括以下步骤：将双功能纳米抗体Nb193-104的编码基因插入pMECS载体，然后导入大肠杆菌WK6感受态细胞，获得重组菌；诱导重组菌表达目的蛋白，裂解重组菌后纯化获得双功能纳米抗体Nb193-104。The content of the present invention also includes the preparation method of the porcine O-type FMDV bifunctional nanobody targeting porcine CD205, comprising the following steps: inserting the coding gene of the bifunctional nanobody Nb193-104 into the pMECS vector, and then introducing it into Escherichia coli WK6 State cells were obtained to obtain recombinant bacteria; the recombinant bacteria were induced to express the target protein, and the recombinant bacteria were lysed and purified to obtain the bifunctional nanobody Nb193-104.
本发明内容还包括靶向猪CD205的猪PEDV双功能纳米抗体，所述双功能纳米抗体以接头元件将编码CD205目的蛋白特异性纳米抗体Nb193和编码猪PEDV抗原特异性纳米抗体Nb2的基因连接后获得获得目的基因片段进一步重组表达获得的目的蛋白。The content of the present invention also includes a porcine PEDV bifunctional nanobody targeting porcine CD205. The bifunctional nanobody uses a linker element to connect the gene encoding the CD205 target protein-specific nanobody Nb193 and the coding porcine PEDV antigen-specific nanobody Nb2 Obtain the target protein obtained by further recombinant expression of the target gene fragment.
其中，所述猪CD205目的蛋白特异性纳米抗体Nb193的核苷酸序列如SEQ ID NO.:9所示，所述猪PEDV抗原特异性纳米抗体Nb2的核苷酸序列如SEQ ID NO.:13所示。Wherein, the nucleotide sequence of the porcine CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.: 9, and the nucleotide sequence of the porcine PEDV antigen-specific nanobody Nb2 is shown in SEQ ID NO.: 13 shown.
其中，所述猪CD205目的蛋白特异性纳米抗体Nb193的氨基酸序列如SEQ ID NO.:11所示，猪PEDV抗原特异性纳米抗体Nb2的氨基酸序列如SEQ ID NO.:14所示。Wherein, the amino acid sequence of the porcine CD205 target protein-specific nanobody Nb193 is shown in SEQ ID NO.:11, and the amino acid sequence of the porcine PEDV antigen-specific nanobody Nb2 is shown in SEQ ID NO.:14.
其中，所述接头元件包括但不仅限于接头元件(G4S)4，还可以包括常见的(G4S)n，n＝1～6，还有驼源抗体IgG2c的铰链区、人抗体IgA的铰链区等，所述接头元件为接头元件(G4S)4，所述接头元件(G4S)4的核苷酸序列如SEQ ID NO.:3所示，接头元件(G4S)4的氨基酸序列如SEQ ID NO.:7所示。Wherein, the linker element includes but not limited to linker element (G4S)4, and may also include common (G4S)n, n=1-6, as well as the hinge region of camel-derived antibody IgG2c, the hinge region of human antibody IgA, etc. , the linker element is a linker element (G4S) 4, the nucleotide sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 3, and the amino acid sequence of the linker element (G4S) 4 is as shown in SEQ ID NO. : As shown in 7.
其中，所述靶向猪CD205的猪PEDV双功能纳米抗体Nb193-2的氨基酸序列如SEQ ID NO.:16所示。其中，所述的猪CD205特异性纳米抗体Nb193与猪CD205目的蛋白的平衡解离常数(K D)为1.04×10 -9，猪PEDV特异性纳米抗体Nb2与猪PEDV抗原的平衡解离常数(K D)为1.03×10 -8，所述的猪CD205/PEDV双功能纳米抗体Nb193-2与猪CD205目的蛋白的平衡解离常数(K D)为1.52×10 -8，所述的猪CD205/PEDV双功 能纳米抗体Nb193-2与猪PEDV抗原的平衡解离常数(K D)为1.01×10 -8。 Wherein, the amino acid sequence of the porcine PEDV bifunctional nanobody Nb193-2 targeting porcine CD205 is shown in SEQ ID NO.:16. Wherein, the equilibrium dissociation constant (K D ) between the porcine CD205-specific nanobody Nb193 and the porcine CD205 target protein is 1.04×10 -9 , and the equilibrium dissociation constant (K D ) between the porcine PEDV-specific nanobody Nb2 and the porcine PEDV antigen ( K D ) is 1.03×10 -8 , the equilibrium dissociation constant (K D ) of the porcine CD205/PEDV bifunctional nanobody Nb193-2 and the target protein of porcine CD205 is 1.52×10 -8 , and the porcine CD205 The equilibrium dissociation constant (K D ) of /PEDV bifunctional nanobody Nb193-2 and porcine PEDV antigen is 1.01×10 -8 .
本发明内容还包括核酸或基因，其编码所述的靶向猪CD205的猪PEDV双功能纳米抗体Nb193-2，其核苷酸序列如SEQ ID NO.:15所示。The content of the present invention also includes nucleic acid or gene, which encodes the porcine PEDV bifunctional nanobody Nb193-2 targeting porcine CD205, the nucleotide sequence of which is shown in SEQ ID NO.:15.
本发明内容还包括所述的靶向猪CD205的猪PEDV双功能纳米抗体的制备方法，包括以下步骤：将双功能纳米抗体Nb193-2的编码基因插入pMECS载体，然后导入大肠杆菌WK6感受态细胞，获得重组菌；诱导重组菌表达目的蛋白，裂解重组菌后纯化获得双功能纳米抗体Nb193-2。The content of the present invention also includes the preparation method of the porcine PEDV bifunctional nanobody targeting porcine CD205, comprising the following steps: inserting the coding gene of the bifunctional nanobody Nb193-2 into the pMECS vector, and then introducing it into Escherichia coli WK6 competent cells , obtain the recombinant bacteria; induce the recombinant bacteria to express the target protein, lyse the recombinant bacteria and purify to obtain the bifunctional nanobody Nb193-2.
本发明内容还包括所述的靶向猪DC细胞的猪O型FMDV的双功能纳米抗体、所述的靶向猪CD205的猪O型FMDV双功能纳米抗体、所述的靶向猪CD205的猪PEDV双功能纳米抗体所述的核酸或基因在制备猪用疫苗中的应用。The contents of the present invention also include the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells, the porcine O-type FMDV bifunctional nanobody targeting porcine CD205, and the porcine CD205-targeting porcine bifunctional nanobody Application of the nucleic acid or gene described in the PEDV bifunctional nanobody in the preparation of a porcine vaccine.
本发明所述载体包括但不仅限于pMECS载体，其他载体，如pHEN1、pHEN4、pComb3XSS、pCANTAB5e、pPIC9K、pYES2等，还有病毒载体，如杆状病毒、慢病毒载体、腺病毒载体、AAV病毒载体、逆转录病毒等，以及转座子和其他基因转移系统，根据后续的表达以及作用方式，可以选择很多种载体形式。The vectors of the present invention include but are not limited to pMECS vectors, other vectors, such as pHEN1, pHEN4, pComb3XSS, pCANTAB5e, pPIC9K, pYES2, etc., and viral vectors, such as baculovirus, lentiviral vector, adenoviral vector, AAV viral vector , Retrovirus, etc., as well as transposons and other gene transfer systems, according to the subsequent expression and mode of action, you can choose a variety of vector forms.
本发明所述细胞包括但不仅限于大肠杆菌WK6感受态细胞，其他宿主细胞如原核细胞中的大肠杆菌TOP10、BL21、XL1-blue等，还有真核细胞CHO、293等，其他宿主还有酿酒酵母BY4743细胞和毕赤酵母GS115细胞，以及sf9昆虫细胞等。The cells of the present invention include but are not limited to Escherichia coli WK6 competent cells, other host cells such as Escherichia coli TOP10, BL21, XL1-blue in prokaryotic cells, eukaryotic cells CHO, 293, etc., and other hosts include brewing Yeast BY4743 cells, Pichia pastoris GS115 cells, and sf9 insect cells, etc.
本发明提供的靶向猪DC细胞的猪O型FMDV双功能纳米抗体具体通过以下技术方案实现：The porcine O-type FMDV bifunctional nanobody targeting porcine DC cells provided by the present invention is specifically realized through the following technical schemes:
1)使用新鲜诱导分化的猪BMDC细胞免疫新疆双峰驼，经过6次免疫后，利用分子生物学原理提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库；1) Use freshly differentiated porcine BMDC cells to immunize Xinjiang Bactrian camels. After 6 times of immunization, use the principles of molecular biology to extract the cDNA of peripheral blood lymphocytes, amplify the antibody heavy chain variable region gene library, and construct phage Display Nanobody gene library;
2)将新鲜诱导分化的猪BMDC细胞作为抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个介导抗原高效内吞的猪DC特异性纳米抗体，命名为Nb131；2) Coat freshly differentiated porcine BMDC cells as antigens, and use phage display technology to perform 3-5 rounds of affinity screening to obtain a porcine DC-specific nanobody that mediates efficient endocytosis of antigens, named Nb131;
3)使用O型FMDV灭活病毒免疫新疆双峰驼，经过6次免疫后，利用分子生物学原理提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库；3) Use O-type FMDV inactivated virus to immunize Bactrian camels in Xinjiang. After 6 times of immunization, use molecular biology principles to extract the cDNA of peripheral blood lymphocytes, amplify the antibody heavy chain variable region gene library, and construct phage display Nanobody gene library;
4)将O型FMDV作为抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个O型FMDV特异性纳米抗体，命名为Nb104；4) Coating O-type FMDV as an antigen, using phage display technology for 3-5 rounds of affinity screening, and obtaining a O-type FMDV-specific nanobody, named Nb104;
5)利用猪DC特异性纳米抗体Nb131和O型FMDV特异性纳米抗体Nb104的基因序列利用重叠延伸PCR方法构建双功能纳米抗体表达体系(Nb131-104)，表达产物经纯化后，与猪O型FMDV抗原孵育，对猪进行免疫，采集免后血清、淋巴结等样品，对抗体、细胞因子进行检测，用于免疫效力评价。5) Using the gene sequences of porcine DC-specific nanobody Nb131 and O-type FMDV-specific nanobody Nb104, a bifunctional nanobody expression system (Nb131-104) was constructed by overlapping extension PCR. After the expression product was purified, it was combined with porcine O-type Incubate with FMDV antigen, immunize pigs, collect samples such as serum and lymph nodes after immunization, and detect antibodies and cytokines for immune efficacy evaluation.
本发明提供的一种靶向猪CD205的猪O型FMDV双功能纳米抗体通过以下技术方案实现：A kind of porcine O-type FMDV bifunctional nanobody targeting porcine CD205 provided by the invention is realized through the following technical solutions:
1)利用PCR技术扩增猪CD205分子的CysR-FNⅡ截短基因序列，采用pET-32a表达载体构建猪CD205分子重组表达载体，进行原核系统可溶性表达及蛋白纯化。1) PCR technology was used to amplify the CysR-FNⅡ truncated gene sequence of porcine CD205 molecule, and pET-32a expression vector was used to construct recombinant expression vector of porcine CD205 molecule, and the soluble expression and protein purification of prokaryotic system were carried out.
2)使用纯化后的猪CD205目的蛋白免疫新疆双峰驼，经过5-7次免疫后提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库。2) Use the purified porcine CD205 target protein to immunize Xinjiang Bactrian camels, extract the cDNA of peripheral blood lymphocytes after 5-7 times of immunization, and amplify the antibody heavy chain variable region gene library to construct phage display nanobody genes library.
3)将纯化后的猪CD205目的蛋白作为抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个高亲和力的猪CD205特异性纳米抗体，命名为Nb193。3) The purified porcine CD205 target protein was coated as an antigen, and phage display technology was used for 3-5 rounds of affinity screening to obtain a high-affinity porcine CD205-specific nanobody, named Nb193.
4)使用猪O型FMDV抗原免疫新疆双峰驼，经过5-7次免疫后提取其外周血淋巴 细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库。4) Use pig O-type FMDV antigen to immunize Xinjiang Bactrian camels, extract the cDNA of peripheral blood lymphocytes after 5-7 times of immunization, and amplify the antibody heavy chain variable region gene library, and construct the phage display nanobody gene library.
5)将猪O型FMDV抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个高亲和力的猪O型FMDV特异性纳米抗体，命名为Nb104。5) The porcine O-type FMDV antigen was coated, and 3-5 rounds of affinity screening were performed using phage display technology to obtain a high-affinity porcine O-type FMDV-specific nanobody, named Nb104.
6)利用猪CD205特异性纳米抗体Nb193和猪O型FMDV特异性纳米抗体Nb104的基因序列经重叠延伸PCR方法构建双功能纳米抗体表达体系(Nb193-104)，表达产物经纯化后，与猪O型FMDV抗原孵育，对猪进行免疫，采集免后血清、淋巴结等样品，对抗体、细胞因子进行检测，用于免疫效力评价。6) The gene sequences of pig CD205-specific nanobody Nb193 and pig O-type FMDV-specific nanobody Nb104 were used to construct a bifunctional nanobody expression system (Nb193-104) by overlapping extension PCR method. After the expression product was purified, it was mixed with pig O Incubate with FMDV antigen, immunize pigs, collect post-immunization serum, lymph nodes and other samples, and detect antibodies and cytokines for immune efficacy evaluation.
本发明提供的靶向猪CD205的猪PEDV双功能纳米抗体按照以下技术方案实现：The porcine PEDV bifunctional nanobody targeting porcine CD205 provided by the present invention is realized according to the following technical scheme:
S1)利用PCR技术扩增猪CD205分子的CysR-FNⅡ截短基因序列，采用pET-32a表达载体构建猪CD205分子重组表达载体，进行原核系统可溶性表达及蛋白纯化。S1) PCR technology was used to amplify the CysR-FNII truncated gene sequence of porcine CD205 molecule, and pET-32a expression vector was used to construct recombinant expression vector of porcine CD205 molecule, and the soluble expression and protein purification of prokaryotic system were carried out.
S2)使用纯化后的猪CD205目的蛋白免疫新疆双峰驼，经过5-7次免疫后提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库。S2) Use the purified porcine CD205 target protein to immunize Xinjiang Bactrian camels, extract the cDNA of peripheral blood lymphocytes after 5-7 times of immunization, and amplify the antibody heavy chain variable region gene library to construct phage display nanobody genes library.
S3)将纯化后的猪CD205目的蛋白作为抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个高亲和力的猪CD205特异性纳米抗体，命名为Nb193。S3) Coating the purified porcine CD205 target protein as an antigen, performing 3-5 rounds of affinity screening using phage display technology, and obtaining a high-affinity porcine CD205-specific nanobody, named Nb193.
S4)使用猪PEDV抗原免疫新疆双峰驼，经过5-7次免疫后提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库。S4) Using porcine PEDV antigen to immunize Bactrian camels in Xinjiang, after 5-7 times of immunization, extract the cDNA of peripheral blood lymphocytes, and amplify the antibody heavy chain variable region gene library, and construct the phage display nanobody gene library.
S5)将猪PEDV抗原进行包被，利用噬菌体展示技术进行3-5轮亲和筛选，获得1个高亲和力的猪PEDV特异性纳米抗体，命名为Nb2。S5) The porcine PEDV antigen was coated, and 3-5 rounds of affinity screening were performed using phage display technology to obtain a high-affinity porcine PEDV-specific nanobody, which was named Nb2.
S6)利用猪CD205特异性纳米抗体Nb193和猪PEDV特异性纳米抗体Nb2的基因序列经重叠延伸PCR方法构建双功能纳米抗体表达体系(Nb193-2)，表达产物经纯化后，与猪PEDV抗原孵育，对猪进行免疫，采集免后血清、淋巴结等样品，对抗体、细胞因子进行检测，用于免疫效力评价。S6) Using the gene sequences of porcine CD205-specific nanobody Nb193 and porcine PEDV-specific nanobody Nb2 to construct a bifunctional nanobody expression system (Nb193-2) by overlapping extension PCR method, the expression product was purified and incubated with porcine PEDV antigen , to immunize pigs, collect post-immunization serum, lymph nodes and other samples, and detect antibodies and cytokines for immune efficacy evaluation.
本发明的靶向猪DC细胞的猪O型FMDV双功能纳米抗体可以同时与猪DC细胞、猪O型口蹄疫抗原特异性结合的双功能纳米抗体，在改善猪O型FMDV抗原免疫效力中开展的应用，可以提高猪O型FMDV抗原免后IgG和IgG1抗体效价，延长免后IgG抗体持续期，提高CD4 +T细胞和CD8 +T细胞占比，促进IFN-γ、IL-2、IL-4分泌。 The porcine O-type FMDV bifunctional nanobody targeting porcine DC cells of the present invention can simultaneously specifically bind porcine DC cells and porcine O-type foot-and-mouth disease antigens, and is carried out in improving the immune efficacy of porcine O-type FMDV antigens Application can increase the titers of IgG and IgG1 antibodies after immunization of pig O-type FMDV antigen, prolong the duration of IgG antibodies after immunization, increase the proportion of CD4 + T cells and CD8 + T cells, and promote IFN-γ, IL-2, IL- 4 secretion.
本发明的靶向猪CD205的猪O型FMDV双功能纳米抗体利用一种可以同时与猪CD205目的蛋白、猪O型FMDV抗原特异性结合的双功能纳米抗体在改善猪O型FMDV抗原免疫效力中开展的应用，可以提高猪O型FMDV抗原免后IgG、IgG1、IgG2a抗体效价，促进淋巴细胞增殖以及IFN-γ、IL-6、IL-4分泌，诱导高效的细胞免疫应答。The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 of the present invention uses a bifunctional nanobody that can specifically bind to porcine CD205 target protein and porcine O-type FMDV antigen at the same time to improve the immune efficacy of porcine O-type FMDV antigen The application carried out can increase the IgG, IgG1, IgG2a antibody titer after immunization with pig O-type FMDV antigen, promote lymphocyte proliferation and IFN-γ, IL-6, IL-4 secretion, and induce efficient cellular immune response.
同样，本发明的靶向猪CD205的猪PEDV双功能纳米抗体利用一种可以同时与猪CD205目的蛋白、猪PEDV抗原特异性结合的双功能纳米抗体在改善猪PEDV抗原免疫效力中开展的应用，可以提高猪PEDV抗原免后IgG、IgG1、IgG2a以及粘膜IgA抗体效价，促进淋巴细胞增殖以及IFN-γ、IL-6、IL-4分泌，诱导高效的细胞免疫应答。Similarly, the porcine CD205-targeted porcine PEDV bifunctional nanobody of the present invention uses a bifunctional nanobody that can specifically bind to porcine CD205 target protein and porcine PEDV antigen at the same time to improve the immune efficacy of porcine PEDV antigen. It can improve the IgG, IgG1, IgG2a and mucosal IgA antibody titers after porcine PEDV antigen immunization, promote lymphocyte proliferation and IFN-γ, IL-6, IL-4 secretion, and induce efficient cellular immune response.
本发明所述双功能纳米抗体可以直接与抗原进行孵育组装，该方法既可以采用全病毒替代蛋白质抗原，充分利用全病毒的天然抗原表位，又可以避免化学偶联和基因融合表达方法对抗原结构的影响，同时，本发明还具有很强的通用性，可以通过更换不同病原特异性的纳米抗体进而构建相应的猪DC靶向或CD205靶向双功能纳米抗体，即可推广应用于其他猪用疫苗抗原。The bifunctional nanobody of the present invention can be incubated and assembled directly with the antigen. This method can not only use the whole virus to replace the protein antigen, make full use of the natural antigen epitope of the whole virus, but also avoid the chemical coupling and gene fusion expression methods. At the same time, the present invention also has strong versatility. By replacing different pathogen-specific nanobodies and then constructing corresponding porcine DC-targeting or CD205-targeting bifunctional nanobodies, it can be extended and applied to other pigs. with vaccine antigens.
有益效果：相对于现有技术，本发明具备以下优点：本发明所提供的可用于抗原靶向猪DC细胞或猪CD205的双功能纳米抗体，是国内外首次提出的以双功能纳米抗体为技术手段，以猪DC细胞或猪CD205分子为靶点探索大幅度改善猪用疫苗抗原免疫效力 的新途径，本发明有助于设计针对猪DC细胞的新型、高效、主动靶向抗原输送系统。本发明采用双功能纳米抗体作为技术纽带，直接与抗原进行孵育组装，即可实现抗原的DC或CD205靶向，该方法既可以采用完整病原粒子替代传统的蛋白质抗原，充分利用完整病原粒子的天然抗原表位，又可以避免常规的化学偶联和基因融合表达方法对抗原结构的影响。此外，本发明具备通用性，可以针对不同的病原筛选其特异性的纳米抗体，构建相应的猪DC靶向型双功能纳米抗体，即可扩展应用于其他猪用疫苗抗原，操作简单、高效、耗时短，且双功能纳米抗体在设计以及结构上较为明确，易于利用微生物基因工程系统高效生产，制造成本低，具有较好的稳定性。双功能纳米抗体与抗原经孵育后即可使用，简单易行，更加贴近兽医临床实际，便于推广普及。Beneficial effects: Compared with the prior art, the present invention has the following advantages: the bifunctional nanobody provided by the present invention, which can be used to target antigens to porcine DC cells or porcine CD205, is the first domestic and foreign bifunctional nanobody technology Means, using porcine DC cells or porcine CD205 molecules as the target to explore a new way to greatly improve the immune efficacy of porcine vaccine antigens, the invention helps to design a new, efficient, and actively targeted antigen delivery system for porcine DC cells. In the present invention, the bifunctional nanobody is used as a technical link to directly incubate and assemble the antigen to realize DC or CD205 targeting of the antigen. This method can replace the traditional protein antigen with complete pathogenic particles, and fully utilize the natural properties of complete pathogenic particles. Antigen epitopes can avoid the influence of conventional chemical coupling and gene fusion expression methods on the antigen structure. In addition, the present invention has versatility, can screen specific nanobodies for different pathogens, and construct corresponding porcine DC-targeting bifunctional nanobodies, which can be extended and applied to other porcine vaccine antigens. The operation is simple, efficient, and efficient. The time-consuming is short, and the design and structure of the bifunctional nanobody are relatively clear, and it is easy to use the microbial genetic engineering system to efficiently produce, the manufacturing cost is low, and it has good stability. The bifunctional nanobody and antigen can be used after incubation, which is simple and easy to implement, closer to veterinary clinical practice, and easy to popularize.
附图说明Description of drawings
图1为激光共聚焦显微镜观察猪BMDC细胞免疫荧光图。分别利用PE anti-porcine CD11c、FITC anti-porcine CD11b和DAPI对细胞进行染色并观察细胞形态。Figure 1 is the immunofluorescence image of porcine BMDC cells observed by laser confocal microscope. The cells were stained with PE anti-porcine CD11c, FITC anti-porcine CD11b and DAPI respectively, and the cell morphology was observed.
图2为VHH基因PCR扩增结果。其中M：DL2000bp DNA marker，泳道1和2为VHH基因片段扩增产物。Fig. 2 is the result of PCR amplification of VHH gene. Among them, M: DL2000bp DNA marker, lanes 1 and 2 are amplification products of VHH gene fragments.
图3为噬菌体基因文库单克隆的鉴定电泳图。其中泳道1-24分别表示随机挑选构建的噬菌体基因文库单克隆，M：DL2000bp DNA marker。Fig. 3 is an electrophoresis diagram of identification of a single clone of a phage gene library. Among them, lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, M: DL2000bp DNA marker.
图4为间接ELISA方法检测纳米抗体的结合活性。横坐标表示不同纳米抗体编号，纵坐标表示OD450数值，猪DC破碎产物表示样品孔，猪骨髓祖细胞破碎产物表示阴性孔，Control表示空白孔。Figure 4 is an indirect ELISA method for detecting the binding activity of Nanobodies. The abscissa indicates different nanobody numbers, the ordinate indicates the OD450 value, the porcine DC fragmentation product represents the sample well, the porcine bone marrow progenitor cell fragmentation product represents the negative well, and Control represents the blank well.
图5为纯化后的纳米抗体的SDS-PAGE电泳图和Western Blot鉴定结果。左图中泳道1-6为随机挑选的纯化后的纳米抗体SDS-PAGE鉴定结果；M:protein standards；右图中泳道1-6为随机挑选的纯化后的纳米抗体Western Blot鉴定结果；M:protein standards。Figure 5 is the SDS-PAGE electrophoresis and Western Blot identification results of the purified Nanobodies. Lanes 1-6 in the left figure are the results of SDS-PAGE identification of randomly selected purified Nanobodies; M: protein standards; lanes 1-6 in the right figure are the results of Western Blot identification of randomly selected purified Nanobodies; M: protein standards.
图6为VHH基因PCR扩增结果。其中M：DL2000bp DNA marker，泳道1-5为VHH基因片段扩增产物。Fig. 6 is the result of PCR amplification of VHH gene. Among them, M: DL2000bp DNA marker, and lanes 1-5 are amplification products of VHH gene fragments.
图7为噬菌体基因文库单克隆的鉴定电泳图。其中泳道1-24分别表示随机挑选构建的噬菌体基因文库单克隆，M：DL2000bp DNA marker。Fig. 7 is an electrophoresis diagram of identification of a single clone of a phage gene library. Among them, lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, M: DL2000bp DNA marker.
图8为间接ELISA方法检测纳米抗体的结合活性。横坐标表示不同纳米抗体编号，纵坐标表示OD450数值，O型FMDV表示样品孔，BHK-21表示阴性孔，Control表示空白孔。Figure 8 is an indirect ELISA method for detecting the binding activity of Nanobodies. The abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, O-type FMDV indicates the sample well, BHK-21 indicates the negative well, and Control indicates the blank well.
图9为间接ELISA方法检测纳米抗体的特异性。横坐标表示不同纳米抗体编号，纵坐标表示OD450数值，A型FMDV和Asia1型FMDV表示样品孔，Control表示空白孔。Fig. 9 shows the specificity of detecting Nanobodies by indirect ELISA method. The abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, type A FMDV and Asia1 type FMDV indicate the sample well, and Control indicates the blank well.
图10为纯化后的纳米抗体的SDS-PAGE电泳图和Western Blot鉴定结果。左图中泳道1-5为随机挑选的纯化后的纳米抗体SDS-PAGE鉴定结果；M:protein standards；右图中泳道1-5为随机挑选的纯化后的纳米抗体Western Blot鉴定结果；M:protein standards。Figure 10 is the SDS-PAGE electrophoresis and Western Blot identification results of the purified Nanobodies. Lanes 1-5 in the left figure are the results of SDS-PAGE identification of randomly selected purified Nanobodies; M: protein standards; lanes 1-5 in the right figure are the results of Western Blot identification of randomly selected purified Nanobodies; M: protein standards.
图11为双功能纳米抗体基因片段SOE-PCR扩增结果。其中M：DL2000bp DNA marker，另一泳道为双功能纳米抗体基因片段扩增产物。Figure 11 is the result of SOE-PCR amplification of the bifunctional Nanobody gene fragment. Among them, M: DL2000bp DNA marker, and the other lane is the amplified product of bifunctional nanobody gene fragment.
图12为纯化后的双功能纳米抗体Nb131-104的SDS-PAGE电泳图和Western Blot鉴定结果。上图中泳道1、2为纯化后的双功能纳米抗体Nb131-104的SDS-PAGE鉴定结果；下图中泳道1、2为纯化后的双功能纳米抗体Nb131-104的Western Blot鉴定结果；M:protein standards。Figure 12 is the SDS-PAGE electropherogram and Western Blot identification results of the purified bifunctional nanobody Nb131-104. Lanes 1 and 2 in the figure above are the SDS-PAGE identification results of the purified bifunctional nanobody Nb131-104; lanes 1 and 2 in the figure below are the Western Blot identification results of the purified bifunctional nanobody Nb131-104; M :protein standards.
图13为激光共聚焦显微镜观察双功能纳米抗体Nb131-104与猪BMDC的结合能力。上方的4组图为加入双功能纳米抗体的试验组激光共聚焦显微镜观察结果，下方的4组图为空白对照组激光共聚焦显微镜观察结果。分别利用AF647 anti-porcine CD1、FITC Nb131-104和DAPI对细胞进行染色并观察细胞形态。Figure 13 shows the binding ability of the bifunctional nanobody Nb131-104 to porcine BMDC observed by confocal laser microscopy. The upper 4 groups of pictures are the results of confocal laser microscope observation of the test group added with bifunctional nanobodies, and the lower 4 groups of pictures are the results of laser confocal microscope observation of the blank control group. The cells were stained with AF647 anti-porcine CD1, FITC Nb131-104 and DAPI, respectively, and the cell morphology was observed.
图14为激光共聚焦显微镜观察双功能纳米抗体Nb131-104的FMDV抗原递送能力。上方的4组图为加入双功能纳米抗体的试验组激光共聚焦显微镜观察结果，下方的4组图为空白对照组激光共聚焦显微镜观察结果。分别利用AF647 anti-porcine CD1、FITC Nb131-104和DAPI对细胞进行染色并观察细胞形态。Fig. 14 shows the FMDV antigen delivery ability of bifunctional nanobody Nb131-104 observed by laser confocal microscope. The upper 4 groups of pictures are the results of confocal laser microscope observation of the test group added with bifunctional nanobodies, and the lower 4 groups of pictures are the results of laser confocal microscope observation of the blank control group. The cells were stained with AF647 anti-porcine CD1, FITC Nb131-104 and DAPI, respectively, and the cell morphology was observed.
图15为ELISA检测免后血清中特异性抗体水平，分别于免后14天、28天、42天、56天采血进行抗体检测。Figure 15 shows the level of specific antibodies in serum after immunization detected by ELISA, and blood was collected on days 14, 28, 42, and 56 after immunization for antibody detection.
图16为ELISA检测免后血清中IgG1抗体水平，分别于免后14天、28天、42天、56天采血进行抗体检测。Figure 16 is the ELISA detection of IgG1 antibody levels in serum after immunization, and blood samples were collected for antibody detection on 14 days, 28 days, 42 days, and 56 days after immunization.
图17 ELISA检测免后血清中IgG2a抗体水平，分别于免后14天、28天、42天、56天采血进行抗体检测。Figure 17 ELISA to detect the level of IgG2a antibody in the serum after immunization, and blood samples were collected on days 14, 28, 42, and 56 after immunization for antibody detection.
图18 ELISA检测免后抗体持续期，分别于免后14天、28天、42天、56天采血进行抗体检测。Figure 18 ELISA to detect the duration of antibodies after immunization, and blood was collected on days 14, 28, 42, and 56 after immunization for antibody detection.
图19 ELISA检测细胞培养上清中IFN-γ、IL-2、IL-4分泌水平；Figure 19 ELISA detection of IFN-γ, IL-2, IL-4 secretion levels in the cell culture supernatant;
图20猪CD205分子CysR-FNⅡ截短基因PCR扩增结果。其中M为DL2000 DNA marker，泳道1为猪CD205分子CysR-FNⅡ截短基因片段扩增产物。Figure 20 PCR amplification results of CysR-FNII truncated gene of porcine CD205 molecule. Among them, M is DL2000 DNA marker, and lane 1 is the amplified product of the CysR-FNII truncated gene fragment of porcine CD205 molecule.
图21纯化后的猪CD205目的蛋白的SDS-PAGE电泳图和Western Blot鉴定结果。其中泳道1为纯化后的猪CD205目的蛋白，M为protein standards。Figure 21 SDS-PAGE electrophoresis and Western Blot identification results of the purified porcine CD205 target protein. Lane 1 is the purified porcine CD205 target protein, and M is protein standards.
图22 VHH基因PCR扩增结果。其中M为DL2000 DNA marker，泳道1和2为VHH基因片段扩增产物。Fig. 22 PCR amplification results of VHH gene. Among them, M is DL2000 DNA marker, and lanes 1 and 2 are amplification products of VHH gene fragments.
图23噬菌体基因文库单克隆的鉴定电泳图。其中泳道1-24分别表示随机挑选构建的噬菌体基因文库单克隆，M为DL2000 DNA marker。Fig. 23 Electropherogram of identification of single clone of phage gene library. Among them, lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, and M is DL2000 DNA marker.
图24间接ELISA方法检测纳米抗体的结合活性。横坐标表示不同纳米抗体编号，纵坐标表示OD450数值，猪CD205目的蛋白表示样品孔，相同条件下制备的其他任一不相关蛋白表示阴性孔，Control表示空白孔。Figure 24 Indirect ELISA method to detect the binding activity of Nanobodies. The abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, the porcine CD205 target protein indicates the sample well, any other irrelevant protein prepared under the same conditions indicates the negative well, and Control indicates the blank well.
图25纯化后的纳米抗体的SDS-PAGE电泳图和Western Blot鉴定结果。泳道1、2为随机挑选的纯化后的纳米抗体，M为protein standards。Figure 25 SDS-PAGE electrophoresis and Western Blot identification results of purified Nanobodies. Lanes 1 and 2 are randomly selected purified nanobodies, and M is protein standards.
图26 VHH基因PCR扩增结果。其中M为DL2000 DNA marker，泳道1-3为VHH基因片段扩增产物。Fig. 26 PCR amplification results of VHH gene. Among them, M is DL2000 DNA marker, and lanes 1-3 are amplification products of VHH gene fragments.
图27噬菌体基因文库单克隆的鉴定电泳图。其中泳道1-24分别表示随机挑选构建的噬菌体基因文库单克隆，M为DL2000 DNA marker。Fig. 27 Electropherogram of identification of single clone of phage gene library. Among them, lanes 1-24 respectively represent the single clones of the phage gene library constructed by random selection, and M is DL2000 DNA marker.
图28间接ELISA方法检测纳米抗体的结合活性。横坐标表示不同纳米抗体编号，纵坐标表示OD450数值，PEDV表示样品孔，ST表示阴性孔，Control表示空白孔。Fig. 28 Indirect ELISA method to detect the binding activity of Nanobodies. The abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, PEDV indicates the sample well, ST indicates the negative well, and Control indicates the blank well.
图29间接ELISA方法检测纳米抗体的特异性。横坐标表示不同纳米抗体编号，纵坐标表示OD450数值，O-FMDV、PCV2、PRRSV、PRV表示样品孔，Control表示空白孔。Figure 29 Indirect ELISA method to detect the specificity of Nanobodies. The abscissa indicates the number of different nanobodies, the ordinate indicates the OD450 value, O-FMDV, PCV2, PRRSV, PRV indicate the sample well, and Control indicates the blank well.
图30纯化后的纳米抗体的SDS-PAGE电泳图和Western Blot鉴定结果。泳道1、2为随机挑选的纯化后的纳米抗体，M为protein standards。Figure 30 SDS-PAGE electrophoresis and Western Blot identification results of purified Nanobodies. Lanes 1 and 2 are randomly selected purified nanobodies, and M is protein standards.
图31双功能纳米抗体基因片段SOE-PCR扩增结果。其中M为DL10000 DNA marker，泳道1为双功能纳米抗体基因片段扩增产物。Fig. 31 SOE-PCR amplification results of bifunctional nanobody gene fragments. Among them, M is DL10000 DNA marker, and lane 1 is the amplified product of bifunctional nanobody gene fragment.
图32纯化后的双功能纳米抗体Nb193-2的SDS-PAGE电泳图和Western Blot鉴定结果。泳道1为纯化后的双功能纳米抗体Nb193-2，M为protein standards。Figure 32 SDS-PAGE electrophoresis and Western Blot identification results of the purified bifunctional nanobody Nb193-2. Lane 1 is the purified bifunctional nanobody Nb193-2, and M is protein standards.
图33激光共聚焦显微镜观察猪BMDC细胞免疫荧光。分别使用PE anti-porcine CD11c、FITC anti-porcine CD11b和DAPI对细胞进行染色并观察细胞形态。Figure 33 Immunofluorescence of porcine BMDC cells observed by laser confocal microscope. The cells were stained with PE anti-porcine CD11c, FITC anti-porcine CD11b and DAPI, respectively, and the cell morphology was observed.
图34激光共聚焦显微镜观察双功能纳米抗体193-2与猪BMDC的结合能力。上方的4组图为加入双功能纳米抗体的试验组激光共聚焦显微镜观察结果，下方的4组图为空白对照组激光共聚焦显微镜观察结果。分别使用AF647 anti-porcine CD1、FITC Nb193-2和DAPI对细胞进行染色并观察细胞形态。Fig. 34 Observation of the binding ability of bifunctional nanobody 193-2 to porcine BMDC by laser confocal microscope. The upper 4 groups of pictures are the results of confocal laser microscope observation of the test group added with bifunctional nanobodies, and the lower 4 groups of pictures are the results of laser confocal microscope observation of the blank control group. The cells were stained with AF647 anti-porcine CD1, FITC Nb193-2 and DAPI, respectively, and the cell morphology was observed.
图35 ELISA检测免后28天血清中特异性抗体水平。Figure 35 ELISA detection of specific antibody levels in serum 28 days after immunization.
图36 ELISA检测免后28天血清中IgG1抗体水平。Figure 36 ELISA detection of IgG1 antibody level in serum 28 days after immunization.
图37 ELISA检测免后28天血清中IgG2a抗体水平。Figure 37 ELISA detection of IgG2a antibody level in serum 28 days after immunization.
图38 ELISA检测免后28天粘膜IgA抗体水平。Figure 38 ELISA detection of mucosal IgA antibody levels 28 days after immunization.
图39 MTT法检测淋巴细胞增殖水平。Figure 39 MTT method to detect the level of lymphocyte proliferation.
图40 ELISA检测细胞培养上清中IFN-γ、IL-6、IL-4分泌水平。Figure 40 ELISA detection of IFN-γ, IL-6, IL-4 secretion levels in the cell culture supernatant.
具体实施方式Detailed ways
本发明结合附图和具体实施例作进一步说明。应该理解，这些实施例仅用于说明目的，而不用于限制本发明范围。The present invention will be further described in conjunction with the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
实施例1 针对猪BMDC细胞纳米抗体文库的构建Example 1 Construction of Nanobody Library against Pig BMDC Cells
1、猪BMDC细胞的诱导分化1. Induced differentiation of porcine BMDC cells
收集新鲜的猪骨髓祖细胞(4×10 6cells/mL)，使用猪重组GM-CSF(20ng/mL，购自上海优宁维公司)和猪重组IL-4(10ng/mL，购自上海优宁维公司)对其进行诱导分化，连续培养7天后，收集猪骨髓源树突状细胞(BMDC)并种植于细胞培养皿中(2×10 6cells/mL)，并用4％多聚甲醛室温固定10min，PBS清洗3遍后置于含0.1％Triton的PBS中37℃处理5min，用PBS清洗3遍后置于封闭液中37℃封闭1h，用PE标记anti-porcine CD11c荧光抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤细胞三遍，使用FITC标记anti-porcine CD11b荧光抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤细胞三遍，使用DAPI(工作液，购自上海碧云天公司)染色10min，PBS洗涤细胞三遍后置于激光共聚焦显微镜下进行观察，如图1所示，获得了较为丰富的猪BMDC细胞。 Collect fresh porcine bone marrow progenitor cells (4×10 6 cells/mL), use porcine recombinant GM-CSF (20ng/mL, purchased from Shanghai Unionwell Company) and porcine recombinant IL-4 (10ng/mL, purchased from Shanghai Unionville Company) to induce differentiation, and after continuous culture for 7 days, porcine bone marrow-derived dendritic cells (BMDC) were collected and planted in cell culture dishes (2×10 6 cells/mL), and treated with 4% paraformaldehyde Fix at room temperature for 10 min, wash with PBS three times, place in PBS containing 0.1% Triton for 5 min at 37°C, wash with PBS for three times, place in blocking solution for 1 h at 37°C, and use PE-labeled anti-porcine CD11c fluorescent antibody (1 Incubate at 4°C for 30 min at 4°C, wash cells three times with PBS, and incubate for 30 min at 4°C with FITC-labeled anti-porcine CD11b fluorescent antibody (diluted at 1:1000, purchased from Shanghai Univ Company). , the cells were washed three times with PBS, stained with DAPI (working solution, purchased from Shanghai Biyuntian Company) for 10 min, washed with PBS three times and placed under a confocal laser microscope for observation, as shown in Figure 1, a relatively abundant Pig BMDC cells.
2、RNA的提取与cDNA的合成2. RNA extraction and cDNA synthesis
经反复多次的诱导分化，将足量的猪BMDC细胞(约1×10 10cells)进行超声破碎，12000g离心10min后弃上清，使用生理盐水重悬沉淀，获得猪BMDC细胞破碎产物，将1mg猪BMDC细胞破碎产物与弗氏完全佐剂等体积混合，对一只新疆双峰驼进行免疫；1周之后，将1mg猪BMDC细胞破碎产物与弗氏不完全佐剂等体积混合，免疫该双峰驼，每周一次，共免疫6次，刺激机体产生针对猪BMDC细胞的特异性抗体；免疫结束后，抽取100mL骆驼外周血淋巴细胞，提取淋巴细胞的总RNA，按照反转录试剂盒(购自TAKARA公司)说明操作，合成cDNA。 After repeated induction and differentiation for many times, a sufficient amount of porcine BMDC cells (about 1×10 10 cells) were ultrasonically disrupted, centrifuged at 12,000 g for 10 min, discarded the supernatant, and resuspended the pellet with normal saline to obtain porcine BMDC cell disruption products. 1 mg of porcine BMDC cell crushing product was mixed with Freund's complete adjuvant in equal volume to immunize a Xinjiang Bactrian camel; 1 week later, 1 mg of porcine BMDC cell crushing product was mixed with Freund's incomplete adjuvant in equal volume to immunize the camel Bactrian camels were immunized once a week for a total of 6 times to stimulate the body to produce specific antibodies against porcine BMDC cells; after the immunization, 100mL camel peripheral blood lymphocytes were extracted to extract the total RNA of lymphocytes, followed by the reverse transcription kit (purchased from TAKARA Company) described the operation and synthesized cDNA.
3、引物的设计与合成3. Design and synthesis of primers
根据参考文献(Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae，Conrath Katja et.al，Antimicrobial Agents and Chemotherapy，2001，45,2807-2812.)设计用于扩增骆驼重链抗体可变区基因VHH片段(350bp)的PCR引物C1F、C1R、V HHF和V HHR。各引物的具体序列如表1。 According to references (Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae, Conrath Katja et.al, Antimicrobial Agents and Chemotherapy, 2001, 45, 2807-2812.) designed to amplify camelid heavy chain antibodies can PCR primers C1F, C1R, VHH F and VHH R of the variable region gene VHH fragment (350bp). The specific sequences of each primer are listed in Table 1.
表1、PCR扩增引物Table 1. PCR amplification primers
Figure PCTCN2021126429-appb-000001
Figure PCTCN2021126429-appb-000001
注：表1中的简并碱基R＝A或G。Note: The degenerate base R=A or G in Table 1.
4.VHH片段的扩增4. Amplification of VHH fragments
利用前面合成的骆驼cDNA为模板，PCR扩增VHH片段。首先，以cDNA为模板，利用C1F和C1R为上下游引物，扩增获得大小约为750bp的基因片段，反应条件为：95℃3min；95℃30s，59℃1min，72℃1min，共30个循环；72℃10min。反应结束后，回收大小约为750bp的基因片段。然后，以该大小约为750bp的基因片段为模板，利用V HHF和V HHR为上下游引物，扩增VHH片段，反应条件为：95℃3min；95℃30s，58℃1min，72℃30s，共30个循环；72℃10min。反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳进行鉴定，紫外灯下观察目的条带，如图2所示，可见大约350bp的VHH基因片段，与预期大小一致。利用凝胶回收试剂盒(购自TAKARA公司)对目的条带进行纯化和回收。 Using the previously synthesized camel cDNA as a template, the VHH fragment was amplified by PCR. First, using cDNA as a template, using C1F and C1R as upstream and downstream primers, amplified gene fragments with a size of about 750bp, the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, and 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered. Then, use the gene fragment with a size of about 750bp as a template, and use V HHF and V HHR as upstream and downstream primers to amplify the VHH fragment. The reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C. After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 2, a VHH gene fragment of about 350 bp was seen, which was consistent with the expected size. The target band was purified and recovered using a gel recovery kit (purchased from TAKARA).
5.噬菌体展示基因文库的构建5. Construction of phage display gene library
纯化回收的VHH基因片段采用Pst I和Not I酶切后，连入pMECS载体(购自Novagen公司)。将连接产物转化至E.coli TG1感受态细胞(购自Novagen公司)中，37℃培养1h，将菌液离心浓缩后涂在含有氨苄抗性的LB平板培养基上，37℃过夜生长之后随机挑选24个单克隆菌落，利用V HHF和V HHR为上下游引物进行菌落PCR鉴定。结果如图3，24个单克隆菌落经菌落PCR鉴定，所有单克隆菌落均含有大小约为350bp的目的片段，说明该文库的插入率达到了100％。将上述平板中的菌落刮入LB液体培养基中，然后加入终浓度为30％的甘油，分装保存于-80℃备用，此即为猪BMDC细胞纳米抗体噬菌体展示文库。 The purified and recovered VHH gene fragment was digested with Pst I and Not I, and then ligated into pMECS vector (purchased from Novagen). The ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers. The results are shown in Figure 3, 24 monoclonal colonies were identified by colony PCR, and all monoclonal colonies contained the target fragment with a size of about 350bp, indicating that the insertion rate of the library reached 100%. Scrape the colonies in the above plate into LB liquid medium, then add glycerol with a final concentration of 30%, and store in -80°C for later use. This is the porcine BMDC cell nanobody phage display library.
实施例2 针对猪BMDC细胞纳米抗体的筛选过程Example 2 Screening process for pig BMDC cell nanobody
1、噬菌体展示文库的扩增1. Amplification of phage display library
取200μL于-80℃冻存的实施例1所制备的噬菌体展示文库，接种至500mL 2×TY培养基中，37℃、摇床转速为200rpm条件下培养3-5h后，加入50μL辅助噬菌体VCSM13(购自Novagen公司)，37℃孵育1h，随后37℃、摇床转速为200rpm条件下培养过夜。次日，加入80g的PEG6000(购自上海生工公司)沉淀噬菌体，该沉淀即为扩增后的噬菌体展示文库。将扩增后的噬菌体展示文库重悬于5mL 0.1M PBS缓冲液中，得到其悬液。Take 200 μL of the phage display library prepared in Example 1 that was frozen at -80°C and inoculate it into 500 mL of 2×TY medium. After culturing for 3-5 hours at 37°C with a shaker speed of 200 rpm, add 50 μL of helper phage VCSM13 (purchased from Novagen), incubated at 37° C. for 1 h, and then cultured overnight at 37° C. with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified phage display library. The amplified phage display library was resuspended in 5mL 0.1M PBS buffer to obtain its suspension.
2、亲和筛选2. Affinity screening
将10μg实施例1制备的猪BMDC细胞破碎产物加入到10mL、100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入96孔酶标板的每孔中，在4℃包被过夜，设立无抗原包被的空白组作为空白对照；次日，每孔加入1％脱脂乳溶液100μL，室温封闭2h；然后，每孔加入100μL扩增后的噬菌体展示文库悬液，室温作用1h，用含有0.05％Tween-20的PBS缓冲液洗5遍，洗掉不结合的噬菌体，随后用100μL浓度为100mM的三乙胺(购自上海生工公司)溶液将与猪BMDC细胞破碎产物特异性结合的噬菌体洗脱下，并感染5倍体积(约500μL)处于对数生长期(OD 600为0.8)的大肠杆菌TG1细胞，37℃培养1h，加入50μL辅助噬菌体VCSM13(购自Novagen公司)侵 染TG1细胞，离心取上清液，即得第一轮筛选到的噬菌体，用于下一轮的筛选。相同筛选过程共进行3轮。每一轮筛选获得的噬菌体各取10μL涂于LB固体培养基上，均在37℃过夜培养，用于观察亲和筛选富集过程。如表2所示，文库经三轮亲和筛选后，每一轮筛选富集到的噬菌体均较上一轮要多。 Add 10 μg of the porcine BMDC cell disruption product prepared in Example 1 to 10 mL, 100 mM NaHCO 3 solution (pH 8.2), mix well, take 100 μL and add it to each well of a 96-well ELISA plate, and coat at 4°C overnight , set up a blank group without antigen coating as a blank control; the next day, add 100 μL of 1% skim milk solution to each well, and block at room temperature for 2 hours; then, add 100 μL of amplified phage display library suspension to each well, and act for 1 hour at room temperature Wash 5 times with PBS buffer containing 0.05% Tween-20 to wash off unbound phages, and then use 100 μL of 100 mM triethylamine (purchased from Shanghai Shenggong Company) solution to specifically dissolve the broken product of porcine BMDC cells. The bound phage was eluted, and infected with 5 times the volume (about 500 μL) of Escherichia coli TG1 cells in the logarithmic growth phase (OD 600 of 0.8), cultured at 37°C for 1 h, and added 50 μL of helper phage VCSM13 (purchased from Novagen) to invade TG1 cells were transfected, and the supernatant was obtained by centrifugation to obtain the phages selected in the first round, which were used for the next round of screening. A total of 3 rounds of the same screening process were carried out. 10 μL of the phages obtained in each round of screening were applied to LB solid medium and cultured overnight at 37°C for observing the enrichment process of affinity screening. As shown in Table 2, after three rounds of affinity screening of the library, more phages were enriched in each round of screening than in the previous round.
表2、噬菌体文库3轮亲和筛选富集过程Table 2. Enrichment process of three rounds of affinity screening of phage library
亲和筛选轮次Affinity screening rounds 投入噬菌体文库量(pfu/mL)Amount of phage library input (pfu/mL) 回收噬菌体文库量(pfu/mL)Amount of recovered phage library (pfu/mL)
第一轮亲和筛选First round of affinity screening 1.12×10 7 1.12×10 7 8.71×10 3 8.71×10 3
第二轮亲和筛选Second round of affinity screening 1.04×10 7 1.04×10 7 6.25×10 4 6.25×10 4
第三轮亲和筛选The third round of affinity screening 1.16×10 7 1.16×10 7 3.04×10 5 3.04×10 5
实施例3 酶联免疫方法(ELISA)筛选特异性阳性克隆Example 3 Enzyme-linked immunosorbent assay (ELISA) screening specific positive clones
1、纳米抗体的表达1. Expression of nanobodies
从实施例2中第三轮筛选后富集在LB平板上的菌落中，挑选200个单菌落分别接种于96孔板(添加有含100μg/mL氨苄青霉素的TB培养基)的各孔中，并设置一个仅加入了TB培养基的空白对照，在37℃、摇床转速为200rpm条件下培养至对数生长期，各孔均加入终浓度为1mM的IPTG，在28℃、摇床转速为200rpm条件下过夜培养。次日，利用超声破碎法裂解各菌，离心后取裂解液，分别获得各重组菌表达的针对猪BMDC细胞破碎产物的纳米抗体，各纳米抗体编号依次为1～200。From the bacterium colonies enriched on the LB plate after the third round of screening in Example 2, pick 200 single colonies and inoculate them in each well of a 96-well plate (with the addition of TB medium containing 100 μg/mL ampicillin), And set up a blank control that only added TB medium, cultivated to the logarithmic growth phase under the conditions of 37°C and shaker speed of 200rpm, each well was added with a final concentration of 1mM IPTG, at 28°C, shaker speed of Cultivate overnight at 200 rpm. On the next day, the bacteria were lysed by ultrasonication, and the lysate was taken after centrifugation to obtain the nanobodies expressed by the recombinant bacteria against the crushed products of porcine BMDC cells. The numbers of each nanobody were 1-200.
2、间接ELISA方法检测纳米抗体的结合活性2. Indirect ELISA method to detect the binding activity of nanobodies
采用间接ELISA反应鉴定编号1～200的各纳米抗体与猪BMDC细胞破碎产物的结合活性。将10μg实施例1制备的猪BMDC细胞破碎产物加入到10mL浓度为100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入到96孔酶标板的每个样品孔中，在4℃包被过夜，对照孔中以猪骨髓祖细胞破碎产物替代猪BMDC细胞破碎产物进行包被；次日，弃掉板内液体，利用含有0.05％Tween-20的PBS缓冲液洗5遍，拍干，每孔加入5％脱脂乳溶液100μL，室温封闭2h；利用含有0.05％Tween-20的PBS缓冲液洗5遍，将100μL各纳米抗体依次加入ELISA板的各孔中，室温下孵育1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的纳米抗体，加入100μL经1:2000稀释后的Mouse anti-HA tag antibody(鼠抗HA抗体，购自北京康为世纪公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入100μL经1:2000稀释后的HRP labeled goat anti-mouse IgG(辣根过氧化物酶标记的山羊抗小鼠抗体，购自艾美捷公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入辣根过氧化物酶显色液(购自上海生工公司)，37℃孵育15min，每孔各加2M的硫酸溶液50μL终止反应，使用酶标仪测定各孔在450nm波长处的吸光值OD 450。当样品孔OD 450值大于对照孔OD 450值2.5倍以上时，判为阳性克隆孔。结果如图4所示，共有17个纳米抗体可与猪BMDC细胞破碎产物发生特异性的结合反应(为筛选到高亲和力的纳米抗体，仅选取OD 450值大于2.0的纳米抗体)。 The indirect ELISA reaction was used to identify the binding activity of each Nanobody numbered 1-200 to the broken product of porcine BMDC cells. The porcine BMDC cell disruption product that 10 μ g embodiment 1 prepares joins 10mL concentration and is the NaHCO of 100mM in the solution (pH8.2), mixes homogeneously, gets 100 μ L and joins in each sample hole of 96-well microtiter plate, in 4 ℃ for overnight coating, and in the control wells, the broken product of porcine bone marrow progenitor cells was used to replace the broken product of porcine BMDC cells for coating; the next day, discard the liquid in the plate, wash 5 times with PBS buffer containing 0.05% Tween-20, and shoot Dry, add 100 μL of 5% skim milk solution to each well, block at room temperature for 2 h; wash 5 times with PBS buffer containing 0.05% Tween-20, add 100 μL of each nanobody to each well of the ELISA plate in turn, incubate at room temperature for 1 h, Use PBS buffer containing 0.05% Tween-20 to wash away unbound nanobodies, add 100 μL of Mouse anti-HA tag antibody (mouse anti-HA antibody, purchased from Beijing Kangwei Century Company) diluted 1:2000, and keep at room temperature Leave it for 1 hour, wash unbound antibody with PBS buffer containing 0.05% Tween-20, add 100 μL of HRP labeled goat anti-mouse IgG (horseradish peroxidase-labeled goat anti-mouse IgG) diluted 1:2000 Antibodies (purchased from Amethyst) were placed at room temperature for 1 h, unbound antibodies were washed away with PBS buffer containing 0.05% Tween-20, and horseradish peroxidase chromogenic solution (purchased from Shanghai Sangon) was added. , incubate at 37°C for 15 min, add 50 μL of 2M sulfuric acid solution to each well to stop the reaction, and use a microplate reader to measure the absorbance value OD 450 of each well at a wavelength of 450 nm. When the OD 450 value of the sample well is more than 2.5 times greater than the OD 450 value of the control well, it is judged as a positive clone well. The results are shown in Figure 4, a total of 17 nanobodies can specifically bind to the broken product of porcine BMDC cells (in order to screen high-affinity nanobodies, only nanobodies with an OD value greater than 2.0 were selected).
实施例4 表面等离子共振法(SPR)筛选针对猪BMDC的高亲和力纳米抗体Example 4 Surface plasmon resonance (SPR) screening for high-affinity nanobodies against porcine BMDC
1、针对猪BMDC的纳米抗体的表达及纯化1. Expression and purification of nanobodies against porcine BMDC
分别抽提实施例3所获得的针对猪BMDC的表达阳性纳米抗体的重组菌质粒，42℃分别转化到大肠杆菌WK6感受态细胞(购自Novagen公司)中，37℃、摇床转速为200rpm的条件下培养1h，离心浓缩菌液，并将其涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16小时；挑选单个菌落，分别获得针对猪BMDC的表达纳米抗体的重组菌A1-A17。The recombinant bacterial plasmids expressing positive Nanobodies against porcine BMDCs obtained in Example 3 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42° C. Cultivate under conditions for 1 hour, centrifuge and concentrate the bacterial solution, spread it on an LB plate containing 100 μg/mL ampicillin, and incubate at 37°C for 12 to 16 hours; pick a single colony, and obtain recombinants expressing nanobodies targeting porcine BMDC Bacteria A1-A17.
将重组菌A1-A17分别接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600达0.6～0.9，取1mL菌液转接至500mL TB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1mM的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎获得纳米抗体粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法纯化纳米抗体。随机取纯化后的纳米抗体(编号分别为18、29、37、47、60、64)进行SDS-PAGE电泳和Western blot鉴定。从如图5可以看到纳米抗体在大约16kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 Inoculate the recombinant bacteria A1-A17 in 5 mL of LB culture solution containing ampicillin, and culture them in a shaker at 37°C until the OD 600 reaches 0.6-0.9. Cultivate in a shaker. When the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1mM, and culture in a shaker at 28°C for 12-16 hours to induce the recombinant bacteria to express the target protein, collect the bacterial precipitate by centrifugation, and use ultrasonic crushing The nanobody crude extract was obtained, and the nanobody was purified by affinity chromatography using a nickel column (purchased from GE Healthcare). Purified nanobodies (numbered 18, 29, 37, 47, 60, 64) were randomly selected for SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 5 that the nanobodies have obvious bands at about 16kD, consistent with the expected size of the target fragment, and the purity is over 90%.
2、SPR法鉴定针对猪BMDC的纳米抗体的亲和力2. Identification of the affinity of nanobodies against porcine BMDC by SPR method
分别使用Biacore TM X100蛋白互作仪(购自GE Healthcare公司)鉴定所获得的17个针对猪BMDC的纳米抗体与猪BMDC细胞破碎产物的亲和力。首先使用偶联试剂N-ethyl-N’-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide(购自Sigma公司)将实施例1制备的1mg猪BMDC细胞破碎产物偶联至CM5芯片上，利用生理盐水将纯化后的纳米抗体(编号如表3所示)从100nM进行梯度稀释(分别为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM)，并分别与偶联至CM5芯片上的破碎产物进行结合，结合时间180s，使用乙醇胺进行封闭，利用HBS-EP缓冲液(10mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid(HEPES)，pH 7.5，150mM NaCl,3.5mM EDTA,and 0.005％(v/v)Surfactant P20)进行洗涤去除未结合的物质，流速为30μL/min，使用10mM glycine/HCl(pH 2.5)进行重生。检测结果经Biacore 2.0.1软件进行分析，结合速率常数(ka)、解离速率常数(kd)以及平衡解离常数(K D)如表3所示。编号为131的纳米抗体K D值达3.43×10 -9，为本发明筛选获得的亲和力最高的针对猪BMDC的纳米抗体，后续研究均围绕该纳米抗体开展。 The affinities of the obtained 17 nanobodies against porcine BMDCs and the disrupted products of porcine BMDCs were identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare). First, use the coupling reagent N-ethyl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma) to couple the 1 mg porcine BMDC cell breakdown product prepared in Example 1 to the CM5 chip, and use physiological saline The purified nanobodies (numbered as shown in Table 3) were serially diluted from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and respectively combined with the fragmented products coupled to the CM5 chip For binding, the binding time is 180s, and ethanolamine is used for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v/v) Surfactant P20) to remove unbound material by washing at a flow rate of 30 μL/min and regenerate using 10 mM glycine/HCl (pH 2.5). The detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 3. The nanobody numbered 131 has a K D value of 3.43×10 -9 , which is the nanobody with the highest affinity for porcine BMDC obtained through the screening of the present invention. Subsequent studies will be carried out around this nanobody.
表3.各纳米抗体与猪BMDC细胞破碎产物结合的动力学参数Table 3. Kinetic parameters of each Nanobody binding to porcine BMDC cell breakdown products
纳米抗体编号Nanobody ID k a(M -1s -1) k a (M -1 s -1 ) k d(s -1) k d (s -1 ) K D(M) K D (M)
1818 3.16×10 4 3.16×10 4 4.10×10 -4 4.10×10 -4 1.29×10 -8 1.29×10 -8
2929 1.17×10 4 1.17×10 4 3.97×10 -4 3.97×10 -4 3.39×10 -8 3.39×10 -8
3737 4.11×10 4 4.11×10 4 3.03×10 -4 3.03×10 -4 0.73×10 -8 0.73×10 -8
3939 8.85×10 4 8.85×10 4 1.52×10 -4 1.52×10 -4 0.17×10 -8 0.17×10 -8
4747 2.15×10 5 2.15×10 5 9.65×10 -4 9.65×10 -4 4.48×10 -9 4.48×10 -9
6060 1.91×10 4 1.91×10 4 5.05×10 -4 5.05×10 -4 2.64×10 -8 2.64×10 -8
6464 5.18×10 4 5.18×10 4 1.79×10 -4 1.79×10 -4 0.35×10 -8 0.35×10 -8
9696 8.09×10 4 8.09×10 4 4.93×10 -4 4.93×10 -4 0.61×10 -8 0.61×10 -8
108108 7.17×10 4 7.17×10 4 3.18×10 -4 3.18×10 -4 0.44×10 -8 0.44×10 -8
128128 2.10×10 4 2.10×10 4 4.76×10 -4 4.76×10 -4 2.26×10 -8 2.26×10 -8
131131 0.89×10 5 0.89×10 5 3.05×10 -4 3.05×10 -4 3.43×10 -9 3.43×10 -9
147147 6.71×10 4 6.71×10 4 7.32×10 -4 7.32×10 -4 1.09×10 -8 1.09×10 -8
162162 5.45×10 4 5.45×10 4 8.23×10 -4 8.23×10 -4 1.51×10 -8 1.51×10 -8
169169 3.10×10 4 3.10×10 4 2.98×10 -4 2.98×10 -4 0.96×10 -8 0.96×10 -8
173173 9.12×10 4 9.12×10 4 1.76×10 -4 1.76×10 -4 0.19×10 -8 0.19×10 -8
181181 4.46×10 4 4.46×10 4 8.81×10 -4 8.81×10 -4 1.97×10 -8 1.97×10 -8
194194 2.01×10 4 2.01×10 4 5.24×10 -4 5.24×10 -4 2.61×10 -8 2.61×10 -8
注：表3中ka代表结合速率常数，kd代表解离速率常数，K D代表平衡解离常数。 Note: In Table 3, ka represents the association rate constant, kd represents the dissociation rate constant, and K D represents the equilibrium dissociation constant.
抽提纳米抗体Nb131的重组菌质粒，送上海生工公司进行序列测定，获得其核苷酸序列和氨基酸序列，其核苷酸序列和氨基酸序列分别如SEQ ID NO.:1和SEQ ID NO.:5所示。The recombinant bacterial plasmid of nanobody Nb131 was extracted and sent to Shanghai Shenggong Company for sequence determination to obtain its nucleotide sequence and amino acid sequence. The nucleotide sequence and amino acid sequence are respectively shown as SEQ ID NO.:1 and SEQ ID NO. :5 shown.
实施例5 针对O型口蹄疫病毒纳米抗体文库的构建Example 5 Against the construction of O-type foot-and-mouth disease virus nanobody library
1、RNA的提取与cDNA的合成1. RNA extraction and cDNA synthesis
将1mg O型口蹄疫灭活病毒(中农威特生物科技股份有限公司惠赠)与弗氏完全佐剂等体积混合，对一只新疆双峰驼进行免疫；1周之后，将1mg O型口蹄疫灭活病毒与弗氏不完全佐剂等体积混合，免疫该双峰驼，每周一次，共免疫6次，刺激机体产生针对抗原的特异性抗体；免疫结束后，提取100mL骆驼外周血淋巴细胞，提取淋巴细胞的总RNA，按照反转录试剂盒(购自TAKARA公司)说明操作，合成cDNA。Mix 1mg of O-type foot-and-mouth disease inactivated virus (gifted by Zhongnong Weite Biotechnology Co., Ltd.) with Freund's complete adjuvant in equal volumes to immunize a Xinjiang Bactrian camel; 1 mg O-type foot-and-mouth disease inactivated The virus was mixed with an equal volume of Freund's incomplete adjuvant, and the Bactrian camel was immunized once a week for a total of 6 times to stimulate the body to produce specific antibodies against the antigen; after the immunization, 100mL camel peripheral blood lymphocytes were extracted and extracted The total RNA of lymphocytes was operated according to the instructions of the reverse transcription kit (purchased from TAKARA Company) to synthesize cDNA.
2、引物的设计与合成2. Design and synthesis of primers
根据参考文献(Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae，Conrath Katja et.al，Antimicrobial Agents and Chemotherapy，2001，45,2807-2812.)设计用于扩增骆驼重链抗体可变区基因VHH片段(350bp)的PCR引物C1F、C1R、V HHF和V HHR。各引物的具体序列如上述表1。 According to references (Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae, Conrath Katja et.al, Antimicrobial Agents and Chemotherapy, 2001, 45, 2807-2812.) designed to amplify camelid heavy chain antibodies can PCR primers C1F, C1R, VHH F and VHH R of the variable region gene VHH fragment (350bp). The specific sequence of each primer is shown in Table 1 above.
3、VHH片段的扩增3. Amplification of VHH fragments
利用本实施例标题1中合成的骆驼cDNA为模板，PCR扩增VHH片段。首先，以cDNA为模板，利用C1F和C1R为上下游引物，扩增获得大小约为750bp的基因片段，反应条件为：95℃3min；95℃30s，59℃1min，72℃1min，共30个循环；72℃10min。反应结束后，回收大小约为750bp的基因片段。然后，以该大小约为750bp的基因片段为模板，利用V HHF和V HHR为上下游引物，扩增VHH片段，反应条件为：95℃3min；95℃30s，58℃1min，72℃30s，共30个循环；72℃10min。反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳进行鉴定，紫外灯下观察目的条带，如图6所示，可见大约350bp的VHH基因片段，与预期大小一致。利用凝胶回收试剂盒(购自TAKARA公司)说明书进行操作，对目的条带进行纯化和回收。 Using the camel cDNA synthesized in Title 1 of this example as a template, the VHH fragment was amplified by PCR. First, using cDNA as a template, using C1F and C1R as upstream and downstream primers, amplified gene fragments with a size of about 750bp, the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, and 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered. Then, use the gene fragment with a size of about 750bp as a template, and use V HHF and V HHR as upstream and downstream primers to amplify the VHH fragment. The reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C. After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 6, a VHH gene fragment of about 350 bp was seen, which was consistent with the expected size. Purify and recover the target bands by using the instruction manual of the gel recovery kit (purchased from TAKARA company).
4、噬菌体展示基因文库的构建4. Construction of phage display gene library
纯化回收的VHH基因片段采用PstI和NotI酶切后，连入pMESC载体(购自Novagen公司)。将连接产物转化至E.coli TG1感受态细胞(购自Novagen公司)中，37℃培养1h，将菌液离心浓缩后涂在含有氨苄抗性的LB平板培养基上，37℃过夜生长之后随机挑选24个单克隆菌落，利用V HHF和V HHR为上下游引物进行菌落PCR鉴定。结果如图7，24个单克隆菌落经菌落PCR鉴定，所有单克隆菌落均含有大小约为350bp的目的片段，说明该文库的插入率达到了100％。将上述平板中的菌落刮入LB液体培养基中，然后加入终浓度为30％的甘油，分装保存于-80℃备用，此即为O型口蹄疫病毒噬菌体展示文库。 The purified and recovered VHH gene fragment was digested with PstI and NotI, and then ligated into pMESC vector (purchased from Novagen). The ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers. The results are shown in Figure 7, 24 monoclonal colonies were identified by colony PCR, and all monoclonal colonies contained the target fragment with a size of about 350bp, indicating that the insertion rate of the library reached 100%. Scrape the bacterium colonies in the above plate into LB liquid medium, then add glycerol with a final concentration of 30%, aliquot and store at -80°C for later use, this is the type O foot-and-mouth disease virus phage display library.
实施例6 针对O型口蹄疫病毒纳米抗体的筛选过程Example 6 Screening process for O-type foot-and-mouth disease virus nanobody
1、噬菌体展示文库的扩增1. Amplification of phage display library
取200μL于-80℃冻存的实施例5制备的O型口蹄疫病毒噬菌体展示文库，接种至500mL 2×TY培养基中，37℃、摇床转速为200rpm条件下培养3-5h后，加入50μL辅助噬菌体VCSM13(购自Novagen公司)，37℃孵育1h，随后37℃、摇床转速为200rpm条件下培养过夜。次日，加入80g的PEG6000(购自上海生工公司)沉淀噬菌体，该沉淀即为扩增后的O型口蹄疫病毒噬菌体展示文库。将噬菌体文库重悬于5mL 0.1M PBS中，得到其悬液。Take 200 μL of the O-type foot-and-mouth disease virus phage display library prepared in Example 5, which was frozen at -80°C, inoculate it into 500mL 2×TY medium, culture it at 37°C and shaker speed at 200rpm for 3-5h, then add 50μL The helper phage VCSM13 (purchased from Novagen) was incubated at 37° C. for 1 h, and then cultured overnight at 37° C. with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified O-type foot-and-mouth disease virus phage display library. Resuspend the phage library in 5mL 0.1M PBS to obtain its suspension.
2、亲和筛选2. Affinity screening
将10μg O型口蹄疫灭活病毒加入到10mL、100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入96孔酶标板的每孔中，在4℃包被过夜，设立BHK-21细胞破碎产物(本实验室保存)作为对照；次日，每孔加入100μL、1％脱脂乳溶液，室温封闭2h；然后，每孔加入100μL扩增后的噬菌体文库悬液，室温作用1h，用含有0.05％Tween-20的PBS缓冲液洗5遍，洗掉不结合的噬菌体，随后用100μL、100mM的三乙胺(购自上海生工公司)溶液将与O型口蹄疫病毒特异性结合的噬菌体洗脱下，并感染5倍体积(约500μL)处于对数生长期的大肠杆菌TG1细胞(OD 600为0.8)，37℃培养1h，加入50μL辅助噬菌体VCSM13(购自Novagen公司)侵染TG1细胞，离心取上清液，即得第一轮筛选到的噬菌体，用于下一轮的筛选。相同筛选过程共进行3轮。每一轮筛选获得的噬菌体各取10μL涂于LB固体培养基上，均在37℃过夜培养，用于观察亲和筛选富集过程。如表4所示，文库经三轮亲和筛选后，每一轮筛选富集到的噬菌体均较上一轮要多。 Add 10 μg of O-type foot-and-mouth disease inactivated virus into 10 mL, 100 mM NaHCO 3 solution (pH 8.2), mix well, take 100 μ L and add it to each well of a 96-well microtiter plate, and coat overnight at 4 ° C to set up BHK- 21 Cell disruption products (preserved in our laboratory) were used as a control; the next day, 100 μL of 1% skim milk solution was added to each well, and blocked at room temperature for 2 hours; then, 100 μL of amplified phage library suspension was added to each well, and reacted at room temperature for 1 hour. Wash 5 times with PBS buffer containing 0.05% Tween-20 to wash off the non-binding phages, and then use 100 μL, 100 mM triethylamine (purchased from Shanghai Shenggong Company) solution to bind the bacteriophages that specifically bind to type O foot-and-mouth disease virus. The phage was eluted, and infected with 5 times the volume (about 500 μL) of Escherichia coli TG1 cells in logarithmic growth phase (OD 600 is 0.8), cultured at 37°C for 1 hour, and added 50 μL of helper phage VCSM13 (purchased from Novagen) to infect TG1 The cells were centrifuged to take the supernatant, and the phages selected in the first round were obtained for the next round of screening. A total of 3 rounds of the same screening process were carried out. 10 μL of the phages obtained in each round of screening were applied to LB solid medium and cultured overnight at 37°C for observing the enrichment process of affinity screening. As shown in Table 4, after three rounds of affinity screening of the library, more phages were enriched in each round of screening than in the previous round.
表4.噬菌体文库3轮亲和筛选富集过程Table 4. Three rounds of affinity screening enrichment process of phage library
亲和筛选轮次Affinity screening rounds 投入噬菌体文库量(pfu/mL)Amount of phage library input (pfu/mL) 回收噬菌体文库量(pfu/mL)Amount of recovered phage library (pfu/mL)
第一轮亲和筛选First round of affinity screening 1.01×10 7 1.01×10 7 3.96×10 3 3.96×10 3
第二轮亲和筛选Second round of affinity screening 1.12×10 7 1.12×10 7 2.58×10 4 2.58×10 4
第三轮亲和筛选The third round of affinity screening 1.07×10 7 1.07×10 7 1.49×10 5 1.49×10 5
实施例7 酶联免疫方法(ELISA)筛选特异性阳性克隆Example 7 Enzyme-linked immunosorbent assay (ELISA) screening specific positive clones
1、针对O型口蹄疫病毒的纳米抗体的表达1. Expression of nanobodies against type O foot-and-mouth disease virus
从实施例6中第三轮筛选后富集在LB平板上的菌落中，挑选200个单菌落分别接种于添加有TB培养基(含有100μg/mL氨苄青霉素)的96孔板中，并设置一个仅加入了TB培养基的空白对照，在37℃、摇床转速为200rpm条件下培养至对数生长期，各孔均加入终浓度为1mM的IPTG，在28℃、摇床转速为200rpm条件下过夜培养。次日，利用超声破碎法分别获得各重组菌表达的针对O型口蹄疫病毒的纳米抗体，各纳米抗体编号依次为1～200。From the colonies enriched on the LB plate after the third round of screening in Example 6, 200 single colonies were selected and inoculated respectively in 96-well plates supplemented with TB medium (containing 100 μg/mL ampicillin), and a The blank control with only TB medium was cultured to the logarithmic growth phase at 37°C with a shaker speed of 200rpm, and each well was added with a final concentration of 1mM IPTG. Incubate overnight. On the next day, nanobodies against type O foot-and-mouth disease virus expressed by each recombinant bacteria were respectively obtained by sonicating, and the numbers of each nanobody were 1-200 in sequence.
2、间接ELISA方法检测纳米抗体的结合活性2. Indirect ELISA method to detect the binding activity of nanobodies
采用间接ELISA反应鉴定各纳米抗体与O型口蹄疫病毒的结合活性。将10μg O型口蹄疫灭活病毒加入到10mL 100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入到96孔酶标板的每孔中，在4℃包被过夜，设立BHK-21细胞破碎产物作为对照；次日，弃掉板内液体，利用含有0.05％Tween-20的PBS缓冲液洗5遍，拍干，每孔加入100μL、5％脱脂乳溶液，室温封闭2h；利用含有0.05％Tween-20的PBS缓冲液洗5遍，将各纳米抗体依次加入ELISA板的各孔中，室温下孵育1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的纳米抗体，加入100μL经1:2000稀释后的Mouseanti-HA tag antibody(鼠抗HA抗体，购自北京康为世纪公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入100μL经1:2000稀释后的HRP labeled goat anti-mouse IgG(辣根过氧化物酶标记的山羊抗小鼠抗体，购自艾美捷公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入辣根过氧化物酶显色液(购自上海生工公司)，37℃孵育15min，每孔各加50μL、2M的硫酸溶液终止反应，使用酶标仪测定各孔在450nm波长处的吸光值OD 450。当样品孔OD 450值大于对照孔OD 450值2.5倍以上时，判为阳性克隆孔。结果如图8所示，共有29个纳米抗体可与O型口蹄疫病毒发生特异性的结合反应(为筛选到高亲和力的纳米抗体，仅选取OD 450值大于2.0的纳米抗体)。 The indirect ELISA reaction was used to identify the binding activity of each nanobody to type O foot-and-mouth disease virus. Add 10 μg of O-type foot-and-mouth disease inactivated virus to 10 mL of 100 mM NaHCO 3 solution (pH8.2), mix well, take 100 μ L and add it to each well of a 96-well microtiter plate, and coat overnight at 4 ° C to set up BHK- 21 cell disruption products were used as a control; the next day, the liquid in the plate was discarded, washed 5 times with PBS buffer containing 0.05% Tween-20, patted dry, and 100 μL of 5% skim milk solution was added to each well, and blocked at room temperature for 2 hours; Wash 5 times with PBS buffer containing 0.05% Tween-20, add each Nanobody to each well of the ELISA plate in turn, incubate at room temperature for 1 hour, wash off unbound Nanobodies with PBS buffer containing 0.05% Tween-20 100 μL of Mouseanti-HA tag antibody (mouse anti-HA antibody, purchased from Beijing Kangwei Century Co., Ltd.) diluted 1:2000 was added, left at room temperature for 1 h, and unbound unbound Antibody, add 100 μL of HRP labeled goat anti-mouse IgG (horseradish peroxidase-labeled goat anti-mouse antibody, purchased from Amicate) diluted 1:2000, place at room temperature for 1 h, use 0.05% Tween -20 PBS buffer to wash away unbound antibodies, add horseradish peroxidase chromogenic solution (purchased from Shanghai Sangong Company), incubate at 37°C for 15 min, add 50 μL, 2M sulfuric acid solution to each well to terminate the reaction, Use a microplate reader to measure the absorbance value OD 450 of each well at a wavelength of 450 nm. When the OD 450 value of the sample well is more than 2.5 times greater than the OD 450 value of the control well, it is judged as a positive clone well. The results are shown in Figure 8, a total of 29 nanobodies can specifically bind to O-type FMD virus (in order to screen high-affinity nanobodies, only nanobodies with an OD value greater than 2.0 were selected).
3、特异性的鉴定3. Identification of specificity
按照本实施例中标题2所述间接ELISA检测方法，分别检测各纳米抗体与A型、Asia1型口蹄疫病毒之间的交叉反应性，利用酶标仪测定相应OD 450，不同之处在于ELISA板的包被抗原采用A型、Asia1型口蹄疫病毒替代O型口蹄疫病毒。结果如图9所示，本发明所获得的纳米抗体中，有8个纳米抗体对A型口蹄疫病毒存在交叉反应性，9个纳米抗体对Asia1型口蹄疫病毒存在交叉反应性，3个纳米抗体对A型、Asia1型口蹄疫病毒均存在交叉反应性，其余15个纳米抗体对A型、Asia1型口蹄疫病毒交叉反应性极低，说明本发明获得了针对O型口蹄疫病毒的特异性纳米抗体，同时，部分纳米抗体与A型、Asia1型口蹄疫病毒存在交叉反应性。 According to the indirect ELISA detection method described in Title 2 of this example, the cross-reactivity between each nanobody and type A and Asia1 foot-and-mouth disease virus was detected respectively, and the corresponding OD 450 was measured by a microplate reader. The difference is that the ELISA plate For the coating antigen, type A and Asia1 foot-and-mouth disease virus were used instead of type O foot-and-mouth disease virus. The results are shown in Figure 9, among the nanobodies obtained in the present invention, there are 8 nanobodies that have cross-reactivity to type A foot-and-mouth disease virus, 9 nanobodies have cross-reactivity to Asia1 type foot-and-mouth disease virus, and 3 nanobodies have cross-reactivity to type A foot-and-mouth disease virus. Type A and Asia1 type foot-and-mouth disease viruses all have cross-reactivity, and all the other 15 nanobodies have extremely low cross-reactivity to type A and Asia1 type foot-and-mouth disease viruses, indicating that the present invention has obtained specific nanobodies for O-type foot-and-mouth disease viruses, and at the same time, Some nanobodies had cross-reactivity with type A and Asia1 foot-and-mouth disease virus.
实施例8 表面等离子共振法(SPR)筛选针对O型口蹄疫病毒的高亲和力纳米抗体Example 8 Surface plasmon resonance (SPR) screening for high-affinity nanobodies against O-type foot-and-mouth disease virus
1、纳米抗体的表达及纯化1. Expression and purification of nanobodies
分别抽提实施例7所获得的针对O型口蹄疫病毒的特异性纳米抗体重组菌质粒，42℃分别转化到大肠杆菌WK6感受态细胞(购自Novagen公司)中，37℃、摇床转速为200rpm的条件下培养1h，离心浓缩菌液，并将其涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16小时；挑选单个菌落，分别获得15个表达针对O型口蹄疫病毒纳米抗体的重组菌B1～B15。The specific nanobody recombinant bacterial plasmids for O-type foot-and-mouth disease virus obtained in Example 7 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42 ° C, respectively, at 37 ° C and the shaker speed was 200 rpm Cultivate for 1 hour under the condition of 1 h, centrifuge and concentrate the bacterial solution, spread it on the LB plate containing 100 μg/mL ampicillin, and culture at 37 ° C for 12 to 16 hours; pick a single colony, and obtain 15 expression targets for O-type foot-and-mouth disease virus Nanobody recombinant bacteria B1-B15.
将针对O型口蹄疫病毒的纳米抗体的重组菌B1～B15分别接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600达0.6～0.9，取1mL菌液转接至500mL TB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1mM的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎获得纳米抗体粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法纯化纳米抗体。随机取纯化后的纳米抗体(编号分别为26、43、45、82、88)进行SDS-PAGE电泳和Western blot鉴定。从如图10可以看到纳米抗体在大约16kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 Inoculate the recombinant bacteria B1-B15 of nano-antibodies against O-type foot-and-mouth disease virus into 5 mL of LB culture medium containing ampicillin, culture them in a shaker at 37°C until the OD600 reaches 0.6-0.9, and take 1 mL of the bacteria liquid to transfer to Cultivate in 500mL TB culture medium in a shaker at 37°C. When the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1mM, and culture in a shaker at 28°C for 12-16 hours to induce recombinant bacteria to express the target protein. The bacterial cell precipitate was collected by centrifugation, and the nanobody crude extract was obtained by ultrasonic crushing, and the nanobody was purified by affinity chromatography using a nickel column (purchased from GE Healthcare). Purified Nanobodies (numbered 26, 43, 45, 82, 88) were randomly selected for SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 10 that the nanobodies have obvious bands at about 16kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
2、SPR法鉴定纳米抗体的亲和力2. SPR method to identify the affinity of nanobodies
分别使用Biacore TM X100蛋白互作仪(购自GE Healthcare公司)鉴定所获得的15个纳米抗体与O型口蹄疫灭活病毒的亲和力。首先使用偶联试剂N-ethyl-N’-(dimethyl aminopropyl)-carbodiimide/N-hydroxy succinimide(购自Sigma公司)将1mg O型口蹄疫灭活病毒偶联至CM5芯片上，利用生理盐水将纯化后的各纳米抗体(编号如表5所示)从100nM进行梯度稀释(分别为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM)，并分别与偶联至芯片上的破碎产物进行结合，结合时间180s，使用乙醇胺进行封闭，利用HBS-EP缓冲液(10mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid(HEPES),pH 7.5,150mM NaCl,3.5mM EDTA,and 0.005％(v/v)Surfactant P20)进行洗涤去除未结合的物质，流速为30μL/min，使用10mM glycine/HCl(pH 2.5)进行重生。检测结果经Biacore 2.0.1软件进行分析，结合速率常数(ka)、解离速率常数(kd)以及平衡解离常数(K D)如表5所示。编号为104的纳米抗体K D值达6.82×10 -10，为本发明筛选获得的亲和力最高的纳米抗体，后续研究均围绕该纳米抗体开展。 The affinities of the obtained 15 nanobodies to the inactivated type O foot-and-mouth disease virus were identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare). First, use the coupling reagent N-ethyl-N'-(dimethyl aminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma) to couple 1 mg of inactivated FMD virus type O to the CM5 chip, and use normal saline to Each Nanobody (numbered as shown in Table 5) was serially diluted from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and combined with the fragmented products coupled to the chip respectively, Binding time 180s, using ethanolamine for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v/v) Surfactant P20) was washed to remove unbound material at a flow rate of 30 μL/min and regenerated with 10 mM glycine/HCl (pH 2.5). The detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 5. The Nanobody No. 104 has a K D value of 6.82×10 -10 , which is the Nanobody with the highest affinity obtained through the screening of the present invention. Subsequent studies will be carried out around this Nanobody.
表5.各纳米抗体与O型口蹄疫病毒结合的动力学参数Table 5. Kinetic parameters of each Nanobody binding to O-type foot-and-mouth disease virus
纳米抗体编号Nanobody ID k a(M -1s -1) k a (M -1 s -1 ) k d(s -1) k d (s -1 ) K D(M) K D (M)
2626 5.16×10 5 5.16×10 5 9.10×10 -4 9.10×10 -4 1.76×10 -9 1.76×10 -9
4343 2.57×10 5 2.57×10 5 2.03×10 -4 2.03×10 -4 7.89×10 -8 7.89×10 -8
4545 6.31×10 5 6.31×10 5 3.93×10 -4 3.93×10 -4 6.22×10 -8 6.22×10 -8
8282 4.47×10 5 4.47×10 5 8.25×10 -4 8.25×10 -4 1.84×10 -9 1.84×10 -9
8888 1.52×10 5 1.52×10 5 6.59×10 -4 6.59×10 -4 4.33×10 -9 4.33×10 -9
104104 1.41×10 5 1.41×10 5 9.61×10 -5 9.61×10 -5 6.82×10 -10 6.82×10 -10
109109 8.15×10 5 8.15×10 5 7.91×10 -4 7.91×10 -4 9.71×10 -8 9.71×10 -8
119119 3.09×10 5 3.09×10 5 3.90×10 -4 3.90×10 -4 1.26×10 -9 1.26×10 -9
131131 9.14×10 5 9.14×10 5 6.82×10 -4 6.82×10 -4 7.46×10 -8 7.46×10 -8
146146 7.02×10 5 7.02×10 5 6.47×10 -4 6.47×10 -4 9.21×10 -8 9.21×10 -8
163163 5.89×10 5 5.89×10 5 3.31×10 -4 3.31×10 -4 5.62×10 -8 5.62×10 -8
168168 6.16×10 5 6.16×10 5 2.92×10 -4 2.92×10 -4 4.74×10 -8 4.74×10 -8
184184 1.63×10 5 1.63×10 5 2.31×10 -4 2.31×10 -4 1.41×10 -9 1.41×10 -9
186186 2.14×10 5 2.14×10 5 1.85×10 -4 1.85×10 -4 8.64×10 -8 8.64×10 -8
197197 1.92×10 5 1.92×10 5 1.17×10 -4 1.17×10 -4 6.09×10 -8 6.09×10 -8
注：表5中ka代表结合速率常数，kd代表解离速率常数，K D代表平衡解离常数。 Note: In Table 5, ka represents the association rate constant, kd represents the dissociation rate constant, and K D represents the equilibrium dissociation constant.
抽提纳米抗体Nb104的重组菌质粒，送上海生工公司进行序列测定，获得其基因序列和氨基酸序列，其基因序列和氨基酸序列分别如SEQ ID NO.:2和SEQ ID NO.:6所示。The recombinant bacterial plasmid of nanobody Nb104 was extracted and sent to Shanghai Shenggong Company for sequence determination to obtain its gene sequence and amino acid sequence. The gene sequence and amino acid sequence are shown in SEQ ID NO.:2 and SEQ ID NO.:6 respectively .
实施例9 双功能纳米抗体Nb131-104的串联表达及纯化Example 9 Tandem expression and purification of bifunctional nanobody Nb131-104
1、双功能纳米抗体基因片段的构建1. Construction of bifunctional nanobody gene fragments
分别抽提表达猪DC特异性纳米抗体Nb131和表达猪O型口蹄疫病毒特异性纳米抗体Nb104的重组菌质粒，分别利用表6所示的引物NbF、NbLR和NbLF、NbR进行扩增，获得猪DC特异性的纳米抗体Nb131和猪O型口蹄疫病毒特异性的纳米抗体Nb104的基因片段，并进行纯化回收。利用纯化回收的纳米抗体基因片段为模板，以(G4S)4序列为接头元件linker，通过重叠延伸拼接(Splicing by Overlap Extension,SOE)PCR方法构建双功能纳米抗体片段Nb131-104，反应分两步进行：The recombinant bacterial plasmids expressing porcine DC-specific nanobody Nb131 and porcine O-type foot-and-mouth disease virus-specific nanobody Nb104 were extracted respectively, and amplified using the primers NbF, NbLR, NbLF, and NbR shown in Table 6 to obtain porcine DC The gene fragments of the specific nanobody Nb131 and the porcine O-type foot-and-mouth disease virus specific nanobody Nb104 were purified and recovered. Using the purified and recovered nanobody gene fragment as a template and (G4S)4 sequence as a linker element linker, the bifunctional nanobody fragment Nb131-104 was constructed by splicing by Overlap Extension (SOE) PCR method, and the reaction was divided into two steps conduct:
第一步反应是在不加引物的条件下进行，反应条件为：95℃3min；95℃30s，62℃30s，72℃2min，共8个循环；72℃10min；The first step reaction is carried out without primers, and the reaction conditions are: 95°C for 3min; 95°C for 30s, 62°C for 30s, 72°C for 2min, a total of 8 cycles; 72°C for 10min;
第二步反应只需要在原始PCR管内加入引物NbF和NbR即可，反应条件为：95℃3min；95℃30s，55℃30s，72℃2min，共25个循环；72℃10min；The second step of the reaction only needs to add primers NbF and NbR to the original PCR tube. The reaction conditions are: 95°C for 3min; 95°C for 30s, 55°C for 30s, 72°C for 2min, a total of 25 cycles; 72°C for 10min;
反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳鉴定，紫外灯下观察目的条带，如图11所示，可见大约900bp的基因片段，与预期片段大小相一致，随后插入pMECS载体(购自Novagen公司)的Pst I和Xba I酶切位点之间，于42℃转化到大肠杆菌WK6感受态细胞(购自Novagen公司)，37℃、摇床转速为200rpm的条件下培养1h，离心浓缩菌液，并将其涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16小时；挑选单个菌落，得到表达双功能纳米抗体的重组菌C。各引物的具体序列如表6：After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light. As shown in Figure 11, a gene fragment of about 900bp was seen, which was consistent with the expected fragment size, and then inserted into the pMECS vector (purchased from Novagen) between the Pst I and Xba I restriction sites, transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C, and cultured for 1h at 37°C with a shaker speed of 200rpm , concentrate the bacterial solution by centrifugation, spread it on an LB plate containing 100 μg/mL ampicillin, and incubate at 37° C. for 12 to 16 hours; select a single colony to obtain recombinant bacteria C expressing bifunctional nanobodies. The specific sequence of each primer is shown in Table 6:
表6.SOE-PCR扩增引物Table 6. SOE-PCR amplification primers
Figure PCTCN2021126429-appb-000002
Figure PCTCN2021126429-appb-000002
2、双功能纳米抗体基因序列的鉴定2. Identification of bifunctional nanobody gene sequence
抽提表达双功能纳米抗体Nb131-104重组菌C的质粒，送上海生工公司进行序列测定，获得接头元件(G4S)4和双功能纳米抗体Nb131-104的核苷酸序列以及氨基酸序列。接头元件(G4S)4和双功能纳米抗体Nb131-104的核苷酸序列分别如SEQ ID NO.:3和SEQ ID NO.:4所示，接头元件(G4S)4和双功能纳米抗体Nb131-104的氨基酸序列分别如SEQ ID NO.:7和SEQ ID NO.:8所示。The plasmid expressing the recombinant bacteria C of the bifunctional nanobody Nb131-104 was extracted and sent to Shanghai Sangong Company for sequence determination to obtain the nucleotide sequence and amino acid sequence of the linker element (G4S) 4 and the bifunctional nanobody Nb131-104. The nucleotide sequences of the linker element (G4S) 4 and the bifunctional nanobody Nb131-104 are shown in SEQ ID NO.:3 and SEQ ID NO.:4 respectively, and the linker element (G4S) 4 and the bifunctional nanobody Nb131- The amino acid sequences of 104 are shown in SEQ ID NO.:7 and SEQ ID NO.:8 respectively.
3、双功能纳米抗体的表达与纯化鉴定3. Expression, purification and identification of bifunctional nanobodies
利用重组菌制备双功能纳米抗体Nb131-104。具体方法如下：将重组菌C接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600＝0.6～0.9，取1mL菌液转接至500mL TB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1mM的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎菌体，取裂解液作为双功能纳米抗体粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法进行纯化。取纯化后的双功能纳米抗体进行SDS-PAGE电泳和Western blot鉴定。从图12可以看到双功能纳米抗体Nb131-104在大约35kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 The bifunctional nanobody Nb131-104 was prepared by recombinant bacteria. The specific method is as follows: inoculate the recombinant bacteria C in 5 mL of LB culture solution containing ampicillin, and cultivate it in a shaker at 37°C until OD 600 =0.6-0.9, transfer 1 mL of the bacteria solution to 500 mL of TB culture solution, and incubate at 37°C. Cultivate in a shaker at ℃, when the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1mM, culture in a shaker at 28°C for 12-16 hours to induce recombinant bacteria to express the target protein, collect the bacterial precipitate by centrifugation, and use ultrasonic The bacterium was broken, and the lysate was taken as the crude extract of the bifunctional nanobody, which was purified by affinity chromatography using a nickel column (purchased from GE Healthcare). The purified bifunctional nanobodies were subjected to SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 12 that the bifunctional nanobody Nb131-104 has an obvious band at about 35kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
实施例10 表面等离子共振法(SPR)鉴定双功能纳米抗体Nb131-104的亲和力Example 10 Surface plasmon resonance (SPR) identification of the affinity of the bifunctional nanobody Nb131-104
1、SPR法鉴定纳米抗体的亲和力1. SPR method to identify the affinity of nanobodies
使用Biacore TM X100蛋白互作仪(购自GE Healthcare公司)分别鉴定双功能纳米抗体Nb131-104与猪BMDC细胞破碎产物以及O型口蹄疫病毒的亲和力。首先使用偶联试剂N-ethyl-N’-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide(购自Sigma公司)分别将1mg猪BMDC细胞破碎产物和1mg O型口蹄疫病毒偶联至CM5芯片上，利用生理盐水将纯化后的双功能纳米抗体从100nM进行梯度稀释(分别为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM)，并分别与偶联至芯片上的破碎产物进行结合，结合时间180s，使用乙醇胺进行封闭，利用HBS-EP缓冲液(10mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid(HEPES),pH 7.5,150mM NaCl,3.5mM EDTA,and 0.005％(v/v)Surfactant P20)进行洗涤去除未结合的物质，流速为30μL/min，使用10mM glycine/HCl(pH 2.5)进行重生。检测结果经Biacore 2.0.1软件进行分析，结合速率常数(ka)、解离速率常数(kd)以及平衡解离常数(K D)如表7所示。双功能纳米抗体Nb131-104与猪BMDC细胞破碎产物、O型口蹄疫病毒的平衡解离常数(K D)分别为6.02×10 -8、2.41×10 -9。 Biacore TM X100 protein interaction instrument (purchased from GE Healthcare) was used to identify the affinity of the bifunctional nanobody Nb131-104 to the broken product of porcine BMDC cells and type O foot-and-mouth disease virus, respectively. First, 1 mg of porcine BMDC cell breakdown product and 1 mg of O-type foot-and-mouth disease virus were coupled to the CM5 chip using the coupling reagent N-ethyl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma). The purified bifunctional nanobodies were serially diluted from 100nM in normal saline (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM respectively), and combined with the fragmented products coupled to the chip respectively, the binding time 180s, use ethanolamine to block, utilize HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v /v) Surfactant P20) was washed to remove unbound material at a flow rate of 30 μL/min and regenerated with 10 mM glycine/HCl (pH 2.5). The detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 7. The equilibrium dissociation constants (K D ) of bifunctional nanobody Nb131-104 with porcine BMDC cell breakdown product and type O foot-and-mouth disease virus were 6.02×10 -8 and 2.41×10 -9 , respectively.
表7.双功能纳米抗体Nb131-104与抗原结合的动力学参数Table 7. Kinetic parameters of the binding of bifunctional nanobody Nb131-104 to antigen
抗原antigen k a(M -1s -1) k a (M -1 s -1 ) k d(s -1) k d (s -1 ) K D(M) K D (M)
猪BMDC细胞破碎产物Porcine BMDC cell breakdown product 2.37×10 4 2.37×10 4 1.42×10 -3 1.42×10 -3 6.02×10 -8 6.02×10 -8
O型口蹄疫病毒Type O Foot and Mouth Disease Virus 1.93×10 5 1.93×10 5 4.65×10 -4 4.65×10 -4 2.41×10 -9 2.41×10 -9
注：表7中ka代表结合速率常数，kd代表解离速率常数，K D代表平衡解离常数。 Note: In Table 7, ka represents the association rate constant, kd represents the dissociation rate constant, and K D represents the equilibrium dissociation constant.
实施例11 激光共聚焦显微镜技术鉴定双功能纳米抗体Nb131-104的抗原提呈作用Example 11 Identification of Antigen Presentation of Bifunctional Nanobody Nb131-104 by Confocal Laser Microscopy
1、激光共聚焦显微镜技术鉴定双功能纳米抗体Nb131-104与猪BMDC的结合能力1. Identification of the binding ability of bifunctional nanobody Nb131-104 to porcine BMDC by confocal laser microscopy
将实施例9制备的双功能纳米抗体Nb131-104进行柱层析去除内毒素，并使用内毒素检测试剂盒(购自Pyrosate公司，0.25EU/mL)检测其内毒素含量，确保内毒素水平小于0.05EU，并使用FITC荧光染料(购自上海优宁维公司)对其进行标记，超滤(4000g，20min)除去未标记上的多余染料。按照实施例1所述方法诱导分化新鲜的猪BMDC细胞，使用预先用FITC荧光染料标记的双功能纳米抗体Nb131-104(5μg/mL)与猪BMDC细胞(1×10 6cells/mL)4℃孵育30min，收集细胞，并用4％多聚甲醛室温固定 10min，PBS清洗三遍后置于含0.1％Triton的PBS中37℃处理5min，用PBS清洗三遍后置于封闭液中37℃封闭1h，PBS洗涤细胞三遍，使用AF647标记的抗猪CD1抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤细胞三遍，使用DAPI(工作液，购自碧云天公司)染色10min，PBS洗涤细胞三遍后置于激光共聚焦显微镜下进行观察。结果如图13所示，加入双功能纳米抗体Nb131-104的猪BMDC细胞试验组绿色荧光强度高于对照组，表明双功能纳米抗体Nb131-104可以和猪BMDC细胞发生特异性结合。 The bifunctional nanobody Nb131-104 prepared in Example 9 was subjected to column chromatography to remove endotoxin, and an endotoxin detection kit (purchased from Pyrosate, 0.25EU/mL) was used to detect the endotoxin content to ensure that the endotoxin level was less than 0.05 EU, and labeled with FITC fluorescent dye (purchased from Shanghai Univ Company), and ultrafiltered (4000g, 20min) to remove unlabeled excess dye. Induce and differentiate fresh porcine BMDC cells according to the method described in Example 1, using bifunctional nanobody Nb131-104 (5 μg/mL) pre-labeled with FITC fluorescent dye and porcine BMDC cells (1×10 6 cells/mL) at 4°C Incubate for 30 min, collect cells, fix with 4% paraformaldehyde at room temperature for 10 min, wash with PBS three times, place in PBS containing 0.1% Triton, treat at 37°C for 5 min, wash with PBS three times, place in blocking solution for 1h at 37°C , wash the cells three times with PBS, use AF647-labeled anti-pig CD1 antibody (diluted 1:1000, purchased from Shanghai Uniview Company) and incubate at 4°C for 30 min, wash the cells three times with PBS, use DAPI (working solution, purchased from Beyont company) stained for 10 min, the cells were washed three times with PBS and observed under a confocal laser microscope. The results are shown in Figure 13. The green fluorescence intensity of the pig BMDC cell test group added with the bifunctional nanobody Nb131-104 was higher than that of the control group, indicating that the bifunctional nanobody Nb131-104 can specifically bind to the pig BMDC cells.
2.激光共聚焦显微镜技术鉴定双功能纳米抗体Nb131-104的FMDV抗原递送能力2. Identification of FMDV antigen delivery ability of bifunctional nanobody Nb131-104 by confocal laser microscopy
运用蔗糖密度梯度离心技术对O型FMDV灭活抗原进行纯化，并利用SDS-PAGE和Western Blotting方法对纯化产物进行鉴定。按照实施例1所述方法诱导分化新鲜的猪BMDC细胞，使用FITC荧光染料(购自上海优宁维公司)标记的双功能纳米抗体Nb131-104(5μg/mL)与纯化后的FMDV灭活抗原(5μg/mL)4℃孵育30min后，再与猪BMDC细胞(1×10 6cells/mL)4℃孵育30min，收集细胞，并用4％多聚甲醛室温固定10min，PBS清洗三遍后置于含0.1％Triton的PBS中37℃处理5min，用PBS清洗三遍后置于封闭液中37℃封闭1h，PBS洗涤细胞三遍，使用AF647标记的抗猪CD1抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤细胞三遍，使用DAPI(工作液，购自碧云天公司)染色10min，PBS洗涤细胞三遍后置于激光共聚焦显微镜下进行观察。结果如图14所示，加入双功能纳米抗体Nb131-104的猪BMDC细胞试验组绿色荧光强度高于对照组，表明FMDV抗原经过双功能纳米抗体Nb131-104递送至猪BMDC细胞。 The O-type FMDV inactivated antigen was purified by sucrose density gradient centrifugation, and the purified product was identified by SDS-PAGE and Western Blotting. Fresh porcine BMDC cells were induced to differentiate according to the method described in Example 1, and the bifunctional nanobody Nb131-104 (5 μg/mL) labeled with FITC fluorescent dye (purchased from Shanghai Youningwei Company) and the purified FMDV inactivated antigen were used (5μg/mL) at 4°C for 30min, then incubated with porcine BMDC cells (1×10 6 cells/mL) at 4°C for 30min, collected cells, fixed with 4% paraformaldehyde at room temperature for 10min, washed three times with PBS, and placed in Treat in PBS containing 0.1% Triton at 37°C for 5 min, wash with PBS three times, place in blocking solution for 1 h at 37°C, wash cells with PBS three times, use AF647-labeled anti-pig CD1 antibody (1:1000 dilution, purchased from (Shanghai Youningwei Co., Ltd.) was incubated at 4°C for 30 min, the cells were washed three times with PBS, stained with DAPI (working solution, purchased from Biyuntian Company) for 10 min, washed three times with PBS, and placed under a laser confocal microscope for observation. The results are shown in Figure 14. The green fluorescence intensity of the pig BMDC cell test group added with the bifunctional nanobody Nb131-104 was higher than that of the control group, indicating that the FMDV antigen was delivered to the pig BMDC cells through the bifunctional nanobody Nb131-104.
实施例12 双功能纳米抗体Nb131-104提呈FMDV抗原的免疫效力评价Example 12 Evaluation of the immune efficacy of the FMDV antigen presented by the bifunctional nanobody Nb131-104
1.免疫试验1. Immunization test
将实施例9制备的双功能纳米抗体Nb131-104(5μg/mL)与纯化后的FMDV灭活抗原(5μg/mL)4℃孵育60min后，辅以206免疫佐剂，对猪进行免疫实验，每只猪颈部肌肉免疫2mL，并于不同的时间节点(14d、28d、42d、56d)进行采血取样，同时设置双功能纳米抗体Nb131-104配伍PBS对照组、非DC靶向纳米抗体配伍FMDV对照组、非DC靶向纳米抗体配伍PBS对照组、FMDV对照组和PBS空白对照组，每组5只猪。After incubating the bifunctional nanobody Nb131-104 (5 μg/mL) prepared in Example 9 with the purified FMDV inactivated antigen (5 μg/mL) at 4° C. for 60 min, supplemented with 206 immune adjuvant, the pig was immunized. The neck muscle of each pig was immunized with 2mL, and blood samples were collected at different time points (14d, 28d, 42d, 56d). At the same time, the bifunctional nanobody Nb131-104 was compatible with the PBS control group, and the non-DC targeting nanobody was compatible with FMDV Control group, non-DC targeting nanobody compatibility PBS control group, FMDV control group and PBS blank control group, 5 pigs in each group.
FMD液相阻断ELISA抗体检测技术具有良好的敏感性、快速诊断性和可重复性，与攻毒保护具有很好的相关性，现已被大量应用于FMD免疫抗体水平的检测中，FAO和OIE推荐使用液相阻断ELISA方法来对FMD免疫效果进行评价，故本研究使用液相阻断ELISA方法来对FMD免疫效果进行评价。FMD liquid-phase blocking ELISA antibody detection technology has good sensitivity, rapid diagnosis and reproducibility, and has a good correlation with challenge protection. It has been widely used in the detection of FMD immune antibody levels. FAO and OIE recommends the use of liquid-phase blocking ELISA method to evaluate the immune effect of FMD, so this study uses liquid-phase blocking ELISA method to evaluate the immune effect of FMD.
2.免疫效力评价2. Evaluation of immune efficacy
利用FMD液相阻断ELISA抗体检测试剂盒检测免后血清中特异性抗体水平、抗体亚型(IgG1和IgG2a)及免疫持续期，检测步骤均按照试剂盒说明书进行操作。检测结果如图15所示，双功能纳米抗体Nb131-104配伍FMDV试验组的免后抗体效价均明显高于其他试验组和对照组，表明双功能纳米抗体对FMDV抗原免后抗体产生具有促进作用；抗体亚型检测结果表明(如图16，17所示)，双功能纳米抗体Nb131-104配伍FMDV试验组的免后IgG1抗体效价均明显高于其他试验组和对照组，IgG2a抗体效价与其他FMDV试验组差异不显著，表明双功能纳米抗体Nb131-104可以显著提升FMDV抗原免后IgG1抗体水平，对IgG2a抗体水平的提升作用与其他试验组差异不显著；免疫持续期检测结果显示(如图18所示)，双功能纳米抗体Nb131-104配伍FMDV试验组的抗体水平可持续至免后56天，且抗体效价均明显高于其他试验组和对照组，表明 双功能纳米抗体Nb131-104不仅可以显著提升FMDV抗原免后抗体水平，而且免后56天血清中的抗体仍可以维持在一个较高的水平，表明双功能纳米抗体对FMDV抗原免后所引起的体液免疫反应具有显著的促进作用。The FMD liquid-phase blocking ELISA antibody detection kit was used to detect the specific antibody level, antibody subtype (IgG1 and IgG2a) and the duration of immunity in the serum after immunization, and the detection steps were all operated according to the kit instructions. The test results are shown in Figure 15. The antibody titers of the bifunctional nanobody Nb131-104 compatible with FMDV test group were significantly higher than those of other test groups and control groups after immunization, indicating that the bifunctional nanobody can promote the production of antibodies to FMDV antigen after immunization. Effect; Antibody subtype detection results show (as shown in Figure 16, 17), the IgG1 antibody titer of the bifunctional nanobody Nb131-104 compatibility FMDV test group after immunization is all significantly higher than other test groups and control groups, and the IgG2a antibody titer There was no significant difference between the valency and other FMDV test groups, indicating that the bifunctional nanobody Nb131-104 can significantly increase the IgG1 antibody level after immunization with FMDV antigen, and the effect on increasing the IgG2a antibody level was not significantly different from other test groups; the results of the duration of immunity test showed that (As shown in Figure 18), the antibody level of the bifunctional nanobody Nb131-104 compatibility FMDV test group can last up to 56 days after immunization, and the antibody titers are significantly higher than other test groups and control groups, indicating that the bifunctional nanobody Nb131-104 can not only significantly increase the antibody level of FMDV antigen after immunization, but also maintain a high level of antibody in serum 56 days after immunization, indicating that the bifunctional nanobody has a certain effect on the humoral immune response caused by FMDV antigen after immunization. Significant boost.
于注射位点附近活体采集免疫后猪的新鲜淋巴结，制备淋巴结单细胞悬液，向细胞悬液中加入淋巴细胞分离液，进行密度梯度离心(3000g，20min)，吸取淋巴细胞(离心后从上到下一共分为四层，吸取第二层)，用含血清的细胞培养基洗涤3次后接种至96孔板中(1×10 6cells/mL)；对分离的淋巴细胞分别用抗CD4和CD8的荧光抗体进行染色，利用FACS Aria流式细胞仪检测增殖结果，用CellQuest软件获取数据。在前散射光(FSC)与对侧散射光(SSC)的二维散点图中划出淋巴细胞区P1，在P1区内计数10000个细胞，利用多参数流式细胞术分析淋巴细胞的种类。使用CellQuest软件分析CD4+T细胞和CD8+T细胞占所选定P1细胞区内细胞总数的百分比，各组细胞数及百分比见表8，由表8可知，Nb131-104+O-FMDV试验组CD4+T细胞与CD8+T细胞数均有明显的升高，与其余试验组和对照组相比有非常明显的差异，说明双功能纳米抗体对于FMDV抗原免疫后引起的细胞免疫反应具有促进作用。 Fresh lymph nodes of immunized pigs were collected near the injection site, and lymph node single cell suspension was prepared. Lymphocyte separation medium was added to the cell suspension, and density gradient centrifugation (3000g, 20min) was carried out. Divide into four layers, absorb the second layer), wash 3 times with serum-containing cell culture medium and inoculate into 96-well plate (1×10 6 cells/mL); separate lymphocytes with anti-CD4 Stained with CD8 fluorescent antibody, the proliferation results were detected by FACS Aria flow cytometer, and the data were acquired by CellQuest software. Draw the lymphocyte area P1 in the two-dimensional scatter diagram of forward scattered light (FSC) and opposite side scattered light (SSC), count 10,000 cells in the P1 area, and analyze the types of lymphocytes by multi-parameter flow cytometry . Use CellQuest software to analyze the percentage of CD4+T cells and CD8+T cells in the total number of cells in the selected P1 cell area. The number of CD4+T cells and CD8+T cells were significantly increased, which was significantly different from the rest of the test group and the control group, indicating that the bifunctional nanobody can promote the cellular immune response after FMDV antigen immunization .
表8.各试验组脾脏CD4+T细胞和CD8+T细胞数及百分比Table 8. Number and percentage of spleen CD4+T cells and CD8+T cells in each test group
Figure PCTCN2021126429-appb-000003
Figure PCTCN2021126429-appb-000003
将上述新鲜分离的淋巴细胞(2×10 6cells/mL)接种至24孔板中，用纯化后的FMDV灭活抗原(5μg/mL)体外刺激淋巴细胞，置于细胞培养箱中(37℃，含5％CO 2)共同作用120min，收集部分细胞样品并离心取上清液，按照ELISA试剂盒(购自上海优宁维公司)说明对上清液中IFN-γ、IL-2、IL-4的浓度进行检测，结果如图19所示，Nb131-104+O-FMDV试验组IFN-γ、IL-2、IL-4分泌水平均显著高于其余试验组和对照组，进一步说明双功能纳米抗体Nb131-104可以显著提高FMDV抗原免疫后所引起的细胞免疫反应。 Inoculate the above-mentioned freshly isolated lymphocytes (2×10 6 cells/mL) into 24-well plates, stimulate the lymphocytes in vitro with purified FMDV inactivated antigen (5 μg/mL), and place them in a cell culture incubator (37°C , containing 5% CO 2 ) for 120 min, some cell samples were collected and centrifuged to get the supernatant, and the IFN-γ, IL-2, IL The concentration of -4 was detected, and the results are shown in Figure 19. The secretion levels of IFN-γ, IL-2, and IL-4 in the Nb131-104+O-FMDV test group were significantly higher than those of the rest of the test group and the control group, further illustrating that both The functional nanobody Nb131-104 can significantly improve the cellular immune response induced by FMDV antigen immunization.
实施例13 猪CD205分子目的蛋白的制备Example 13 Preparation of porcine CD205 molecular target protein
1.引物的设计与合成1. Design and synthesis of primers
根据NCBI已公布的猪CD205分子的基因序列(登录号为GQ420669.1)，设计用于扩增猪CD205分子CysR-FNⅡ截短基因(参见SEQ ID NO.:17，600bp)的PCR引物F1和R1。各引物的具体序列如表9。According to the gene sequence of porcine CD205 molecules published by NCBI (accession number is GQ420669.1), PCR primers F1 and R1. The specific sequences of each primer are shown in Table 9.
表9.PCR扩增引物Table 9. PCR amplification primers
引物Primer 序列(5’-3’)Sequence (5'-3')
F1F1 GGATCCGAGCTCTAAATGATCACACCACTGAACGAC(下划线为BamH Ⅰ酶切位点) GGATCC GAGCTCTAAATGATCACACCACTGAACGAC (underlined is the BamH Ⅰ restriction site)
R1R1 TTCGAACTCGAGTGAAATATAAGCTTCTTTCCAAGAA(下划线为HindⅢ酶切位点) TTCGAA CTCGAGTGAAATATAAGCTTCTTTCCAAGAA (the underline is the HindⅢ restriction site)
2.猪CD205分子CysR-FNⅡ截短基因的扩增2. Amplification of CysR-FNⅡ truncated gene of porcine CD205 molecule
无菌条件下获取猪脾脏组织，提取脾脏组织的总RNA，按照反转录试剂盒(购自TAKARA公司)说明操作，合成cDNA，利用本实施例标题1所设计的引物F1和R1，进行PCR扩增，获得大小约为600bp的基因片段。PCR反应条件为：95℃3min；95℃30s，59℃1min，72℃1min，共30个循环；72℃10min。反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳进行鉴定，紫外灯下观察目的条带，如图20所示，可见大约600bp的猪CD205分子CysR-FNⅡ截短基因片段，与预期大小一致。利用凝胶回收试剂盒(购自TAKARA公司)对目的条带进行纯化和回收。Obtain porcine spleen tissue under aseptic conditions, extract the total RNA of spleen tissue, operate according to the instructions of the reverse transcription kit (purchased from TAKARA company), synthesize cDNA, and use the primers F1 and R1 designed in Title 1 of this example to perform PCR Amplify to obtain a gene fragment with a size of about 600bp. The PCR reaction conditions were: 95°C for 3min; 95°C for 30s, 59°C for 1min, 72°C for 1min, a total of 30 cycles; 72°C for 10min. After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 20, it can be seen that the CysR-FNII truncated gene fragment of the porcine CD205 molecule of about 600bp is in line with the expected Same size. The target band was purified and recovered using a gel recovery kit (purchased from TAKARA).
3.猪CD205分子目的蛋白的诱导表达与纯化鉴定3. Induced expression and purification identification of porcine CD205 target protein
将纯化回收的猪CD205分子CysR-FNⅡ基因片段采用BamH Ⅰ和Hind Ⅲ酶切后，连入pET-32a载体(购自Novagen公司)。将连接产物转化至E.coli BL21感受态细胞(购自Novagen公司)中，37℃培养1h，将菌液离心浓缩后涂在含有氨苄青霉素抗性的LB平板培养基上，37℃培养12～16小时；挑选单个菌落，获得表达猪CD205目的蛋白的重组菌1。The purified and recovered CysR-FNII gene fragment of porcine CD205 molecule was digested with BamHI and HindIII, and then ligated into pET-32a vector (purchased from Novagen). The ligation product was transformed into E.coli BL21 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial solution was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, and cultured at 37°C for 12～ 16 hours; pick a single colony to obtain recombinant bacterium 1 expressing the target protein of porcine CD205.
利用重组菌1制备猪CD205目的蛋白。具体方法如下：将重组菌1接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600＝0.6～0.9，取1mL菌液转接至500mL LB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1M的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎重组菌1，取菌体裂解液作为猪CD205目的蛋白粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法纯化猪CD205目的蛋白。取纯化后的蛋白进行SDS-PAGE电泳和Western blot鉴定。从图21可以看到猪CD205目的蛋白在大约23kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 The target protein of porcine CD205 was prepared by recombinant strain 1. The specific method is as follows: Inoculate the recombinant bacteria 1 into 5 mL of LB culture solution containing ampicillin, culture it in a shaker at 37°C until OD 600 =0.6-0.9, take 1 mL of the bacteria solution and transfer it to 500 mL of LB culture solution, Cultivate in a shaker at ℃, when the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1M, and culture in a shaker at 28°C for 12-16 hours to induce the recombinant bacteria to express the target protein, collect the bacterial precipitate by centrifugation, and use ultrasonic The recombinant bacterium 1 was crushed, and the lysate of the bacterium was taken as the crude extract of porcine CD205 target protein, and the target porcine CD205 protein was purified by affinity chromatography using a nickel column (purchased from GE Healthcare). The purified protein was identified by SDS-PAGE electrophoresis and Western blot. It can be seen from Figure 21 that the target protein of porcine CD205 has an obvious band at about 23kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
实施例14 针对猪CD205分子纳米抗体文库的构建Example 14 Construction of Nanobody Library against Porcine CD205 Molecule
1.RNA的提取与cDNA的合成1. RNA extraction and cDNA synthesis
取实施例13所制备的猪CD205目的蛋白1mg与弗氏完全佐剂等体积混合，对一只新疆双峰驼进行免疫；1周之后，将1mg猪CD205目的蛋白与弗氏不完全佐剂等体积混合，免疫该双峰驼，每周一次，共免疫6次，刺激机体产生针对猪CD205目的蛋白的特异性抗体；免疫结束后，抽取100mL骆驼外周血淋巴细胞，提取淋巴细胞的总RNA，按照反转录试剂盒(购自TAKARA公司)说明操作，合成cDNA。Take 1 mg of porcine CD205 target protein prepared in Example 13 and mix it with complete Freund's adjuvant in equal volumes to immunize a Xinjiang Bactrian camel; after 1 week, mix 1 mg of porcine CD205 target protein with Freund's incomplete adjuvant The volume was mixed, and the Bactrian camel was immunized once a week for a total of 6 times to stimulate the body to produce specific antibodies against the target protein of porcine CD205; cDNA was synthesized according to the instructions of the reverse transcription kit (purchased from TAKARA).
2.引物的设计与合成2. Design and synthesis of primers
根据参考文献(Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae，Conrath Katja et.al，Antimicrobial Agents and Chemotherapy，2001，45,2807-2812.)设计用于扩增骆驼重链抗体可变区基因VHH片段(350bp)的PCR引物C1F、C1R、V HHF和V HHR。各引物的具体序列如表10。 According to references (Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae, Conrath Katja et.al, Antimicrobial Agents and Chemotherapy, 2001, 45, 2807-2812.) designed to amplify camelid heavy chain antibodies can PCR primers C1F, C1R, VHH F and VHH R of the variable region gene VHH fragment (350bp). The specific sequences of each primer are shown in Table 10.
表10.PCR扩增引物Table 10. PCR amplification primers
引物Primer 序列(5’-3’)Sequence (5'-3')
C1FC1F GTCCTGGCTGCTCTTCTACAAGGGTCCTGGCTGCTCTTCTACAAGG
C1RC1R GGTACGTGCTGTTGAACTGTTCCGGTACGTGCTGTTGAACTGTTCC
V HHF V HH F GATGTGCAG CTGCAGGAGTCTGGRGGAGG(下划线为Pst Ⅰ酶切位点) GATGTGCAG CTGCAG GAGTCTGGRGGAGG (the underline is the restriction site of Pst Ⅰ)
V HHR V HH R CTAGT GCGGCCGCTGAGGAGACGGTGACCTGGGT(下划线为Not Ⅰ酶切位点) CTAGT GCGGCCGC TGAGGAGACGGTGACCTGGGT (the underline is the Not Ⅰ restriction site)
注：表2中的简并碱基R＝A或G。Note: The degenerate base R=A or G in Table 2.
3.VHH片段的扩增3. Amplification of VHH fragments
利用本实施例标题1中合成的骆驼cDNA为模板，PCR扩增VHH片段。首先，以cDNA为模板，利用C1F和C1R为上下游引物，扩增获得大小约为750bp的基因片段， 反应条件为：95℃3min；95℃30s，59℃1min，72℃1min，共30个循环；72℃10min。反应结束后，回收大小约为750bp的基因片段。然后，以该大小约为750bp的基因片段为模板，利用V HHF和V HHR为上下游引物，扩增VHH片段，反应条件为：95℃3min；95℃30s，58℃1min，72℃30s，共30个循环；72℃10min。反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳进行鉴定，紫外灯下观察目的条带，如图22所示，可见大约350bp的VHH基因片段，与预期大小一致。利用凝胶回收试剂盒(购自TAKARA公司)对目的条带进行纯化和回收。 Using the camel cDNA synthesized in Title 1 of this example as a template, the VHH fragment was amplified by PCR. First, using cDNA as a template, using C1F and C1R as upstream and downstream primers, amplified to obtain gene fragments with a size of about 750bp, the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered. Then, use the gene fragment with a size of about 750bp as a template, and use V HHF and V HHR as upstream and downstream primers to amplify the VHH fragment. The reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C. After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 22, a VHH gene fragment of about 350 bp was seen, which was consistent with the expected size. The target band was purified and recovered using a gel recovery kit (purchased from TAKARA).
4.噬菌体展示基因文库的构建4. Construction of phage display gene library
纯化回收的VHH基因片段采用Pst I和Not I酶切后，连入pMECS载体(购自Novagen公司)。将连接产物转化至E.coli TG1感受态细胞(购自Novagen公司)中，37℃培养1h，将菌液离心浓缩后涂在含有氨苄抗性的LB平板培养基上，37℃过夜生长之后随机挑选24个单克隆菌落，利用V HHF和V HHR为上下游引物进行菌落PCR鉴定。结果如图23，24个单克隆菌落经菌落PCR鉴定，所有单克隆菌落均含有大小约为350bp的目的片段，说明该文库的插入率达到了100％。将上述平板中的菌落刮入LB液体培养基中，然后加入终浓度为30％的甘油，分装保存于-80℃备用，此即为猪CD205分子纳米抗体噬菌体展示文库。 The purified and recovered VHH gene fragment was digested with Pst I and Not I, and then ligated into pMECS vector (purchased from Novagen). The ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers. The results are shown in Figure 23. 24 monoclonal colonies were identified by colony PCR, and all monoclonal colonies contained the target fragment with a size of about 350 bp, indicating that the insertion rate of the library reached 100%. Scrape the colonies on the above plate into LB liquid medium, then add glycerol with a final concentration of 30%, and store in -80°C for later use. This is the porcine CD205 molecular nanobody phage display library.
实施例15 针对猪CD205分子纳米抗体的筛选过程Example 15 Screening process against porcine CD205 molecular nanobodies
1.噬菌体展示文库的扩增1. Amplification of phage display library
取200μL于-80℃冻存的实施例14所制备的噬菌体展示文库，接种至500mL 2×TY培养基中，37℃、摇床转速为200rpm条件下培养3-5h后，加入50μL辅助噬菌体VCSM13(购自Novagen公司)，37℃孵育1h，随后37℃、摇床转速为200rpm条件下培养过夜。次日，加入80g的PEG6000(购自上海生工公司)沉淀噬菌体，该沉淀即为扩增后的噬菌体展示文库。将扩增后的噬菌体展示文库重悬于5mL 0.1M PBS缓冲液中，得到其悬液。Take 200 μL of the phage display library prepared in Example 14 that was frozen at -80°C and inoculate it into 500 mL of 2×TY medium. After culturing for 3-5 hours at 37°C with a shaker speed of 200 rpm, add 50 μL of helper phage VCSM13 (purchased from Novagen), incubated at 37° C. for 1 h, and then cultured overnight at 37° C. with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified phage display library. The amplified phage display library was resuspended in 5mL 0.1M PBS buffer to obtain its suspension.
2.亲和筛选2. Affinity screening
将10μg实施例13所制备的猪CD205目的蛋白加入到10mL、100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入96孔酶标板的每孔中，在4℃包被过夜，设立无抗原包被的空白组作为空白对照；次日，每孔加入1％脱脂乳溶液100μL，室温封闭2h；然后，每孔加入100μL扩增后的噬菌体展示文库悬液，室温作用1h，用含有0.05％Tween-20的PBS缓冲液洗5遍，洗掉不结合的噬菌体，随后用100μL浓度为100mM的三乙胺(购自上海生工公司)溶液将与猪CD205目的蛋白特异性结合的噬菌体洗脱下，并感染5倍体积(约500μL)处于对数生长期(OD 600为0.8)的大肠杆菌TG1细胞，37℃培养1h，加入50μL辅助噬菌体VCSM13(购自Novagen公司)侵染TG1细胞，离心取上清液，即得第一轮筛选到的噬菌体，用于下一轮的筛选。相同筛选过程共进行3轮。每一轮筛选获得的噬菌体各取10μL涂于LB固体培养基上，均在37℃过夜培养，用于观察亲和筛选富集过程。如表11所示，文库经三轮亲和筛选后，每一轮筛选富集到的噬菌体均较上一轮要多。 Add 10 μg of the porcine CD205 target protein prepared in Example 13 to 10 mL, 100 mM NaHCO 3 solution (pH 8.2), mix well, take 100 μL and add it to each well of a 96-well ELISA plate, and coat at 4°C overnight , set up a blank group without antigen coating as a blank control; the next day, add 100 μL of 1% skim milk solution to each well, and block at room temperature for 2 hours; then, add 100 μL of amplified phage display library suspension to each well, and act for 1 hour at room temperature Wash 5 times with PBS buffer containing 0.05% Tween-20 to wash off unbound phages, and then use 100 μL of 100 mM triethylamine (purchased from Shanghai Shenggong Company) solution to specifically bind to the porcine CD205 target protein phage was eluted, and infected with 5 times the volume (about 500 μL) of Escherichia coli TG1 cells in the logarithmic growth phase (OD 600 of 0.8), cultured at 37°C for 1 hour, and added 50 μL of helper phage VCSM13 (purchased from Novagen) to infect The TG1 cells were centrifuged to obtain the supernatant, and the phages selected in the first round were obtained for the next round of screening. A total of 3 rounds of the same screening process were carried out. 10 μL of the phages obtained in each round of screening were applied to LB solid medium and cultured overnight at 37°C for observing the enrichment process of affinity screening. As shown in Table 11, after three rounds of affinity screening of the library, more phages were enriched in each round of screening than in the previous round.
表11.噬菌体文库3轮亲和筛选富集过程Table 11. Enrichment process of three rounds of affinity screening of phage library
亲和筛选轮次Affinity screening rounds 投入噬菌体文库量(pfu/mL)Amount of phage library input (pfu/mL) 回收噬菌体文库量(pfu/mL)Amount of recovered phage library (pfu/mL)
第一轮亲和筛选First round of affinity screening 1.1×10 7 1.1×10 7 5.3×10 3 5.3×10 3
第二轮亲和筛选Second round of affinity screening 1.4×10 7 1.4×10 7 2.2×10 4 2.2×10 4
第三轮亲和筛选The third round of affinity screening 1.2×10 7 1.2×10 7 1.6×10 5 1.6×10 5
实施例16 酶联免疫方法(ELISA)筛选特异性阳性克隆Example 16 Enzyme-linked immunosorbent assay (ELISA) screening specific positive clones
1.纳米抗体的表达1. Expression of Nanobodies
从实施例15中第三轮筛选后富集在LB平板上的菌落中，挑选200个单菌落分别接种于96孔板(添加有含100μg/mL氨苄青霉素的TB培养基)的各孔中，并设置一个仅加入了TB培养基的空白对照，在37℃、摇床转速为200rpm条件下培养至对数生长期，各孔均加入终浓度为1mM的IPTG，在28℃、摇床转速为200rpm条件下过夜培养。次日，利用超声破碎法裂解各菌，离心后取裂解液，分别获得各重组菌表达的、针对CD205目的蛋白的纳米抗体，各纳米抗体编号依次为1～200。From among the colonies enriched on the LB plate after the third round of screening in Example 15, 200 single colonies were selected and inoculated in each well of a 96-well plate (with the addition of TB medium containing 100 μg/mL ampicillin), And set up a blank control that only added TB medium, cultivated to the logarithmic growth phase under the conditions of 37°C and shaker speed of 200rpm, each well was added with a final concentration of 1mM IPTG, at 28°C, shaker speed of Cultivate overnight at 200rpm. On the next day, the bacteria were lysed by ultrasonication, and the lysate was taken after centrifugation to obtain nanobodies against the CD205 target protein expressed by each recombinant bacteria, and the number of each nanobody was 1-200.
2.间接ELISA方法检测纳米抗体的结合活性2. Indirect ELISA method to detect the binding activity of nanobodies
采用间接ELISA反应鉴定编号1～200的各纳米抗体与猪CD205目的蛋白的结合活性。将10μg实施例13所制备的猪CD205目的蛋白加入到10mL浓度为100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入到96孔酶标板的每个样品孔中，在4℃包被过夜，对照孔中以相同条件下制备的其他任一不相关蛋白替代猪CD205目的蛋白进行包被；次日，弃掉板内液体，利用含有0.05％Tween-20的PBS缓冲液洗5遍，拍干，每孔加入5％脱脂乳溶液100μL，室温封闭2h；利用含有0.05％Tween-20的PBS缓冲液洗5遍，将100μL各纳米抗体依次加入ELISA板的各孔中，室温下孵育1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的纳米抗体，加入100μL经1:2000稀释后的Mouse anti-HA tag antibody(鼠抗HA抗体，购自北京康为世纪公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入100μL经1:2000稀释后的HRP labeled goat anti-mouse IgG(辣根过氧化物酶标记的山羊抗小鼠抗体，购自艾美捷公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入辣根过氧化物酶显色液(购自上海生工公司)，37℃孵育15min，每孔各加2M的硫酸溶液50μL终止反应，使用酶标仪测定各孔在450nm波长处的吸光值OD 450。当样品孔OD 450值大于对照孔OD 450值2.5倍以上时，判为阳性克隆孔。结果如图24所示，共有14个纳米抗体可与猪CD205目的蛋白发生特异性的结合反应(为筛选到高亲和力的纳米抗体，仅选取OD 450值大于2.0的纳米抗体)。 The indirect ELISA reaction was used to identify the binding activity of the nanobodies numbered 1-200 to the target protein of porcine CD205. Add 10 μg of the porcine CD205 target protein prepared in Example 13 into 10 mL of 100 mM NaHCO solution (pH 8.2), mix well, get 100 μL and add it to each sample well of a 96-well enzyme plate, and add ℃ for overnight coating, and in the control wells, any other irrelevant protein prepared under the same conditions was used to replace the porcine CD205 target protein for coating; the next day, the liquid in the plate was discarded and washed with PBS buffer containing 0.05% Tween-20. 5 times, pat dry, add 100 μL of 5% skim milk solution to each well, block at room temperature for 2 hours; wash 5 times with PBS buffer containing 0.05% Tween-20, add 100 μL of each nanobody to each well of the ELISA plate in turn, and store at room temperature Incubate for 1 h, use PBS buffer containing 0.05% Tween-20 to wash away unbound Nanobodies, add 100 μL of Mouse anti-HA tag antibody diluted 1:2000 (mouse anti-HA antibody, purchased from Beijing Kangwei Century company) at room temperature for 1 h, washed unbound antibody with PBS buffer containing 0.05% Tween-20, added 100 μL of HRP labeled goat anti-mouse IgG (horseradish peroxidase-labeled Goat anti-mouse antibody (purchased from Amicate), placed at room temperature for 1 h, washed unbound antibody with PBS buffer containing 0.05% Tween-20, added horseradish peroxidase chromogenic solution (purchased from Shanghai Sangon Company), incubated at 37°C for 15 min, added 50 μL of 2M sulfuric acid solution to each well to terminate the reaction, and measured the absorbance value OD 450 of each well at a wavelength of 450 nm using a microplate reader. When the OD 450 value of the sample well is more than 2.5 times greater than the OD 450 value of the control well, it is judged as a positive clone well. The results are shown in Figure 24. A total of 14 Nanobodies can specifically bind to the target protein of porcine CD205 (in order to screen for high-affinity Nanobodies, only Nanobodies with an OD 450 value greater than 2.0 were selected).
实施例17 表面等离子共振法(SPR)筛选高亲和力纳米抗体Example 17 Surface plasmon resonance (SPR) screening of high-affinity nanobodies
1.纳米抗体的表达及纯化1. Expression and purification of nanobodies
分别抽提实施例16所获得的表达纳米抗体的重组菌质粒，42℃分别转化到大肠杆菌WK6感受态细胞(购自Novagen公司)中，37℃、摇床转速为200rpm的条件下培养1h，离心浓缩菌液，并将其涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16小时；挑选单个菌落，分别获得表达纳米抗体的重组菌A1-A14。The plasmids of recombinant bacteria expressing nanobodies obtained in Example 16 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C, and cultured at 37°C and a shaker speed of 200rpm for 1h. Concentrate the bacterial solution by centrifugation, spread it on an LB plate containing 100 μg/mL ampicillin, and incubate at 37°C for 12-16 hours; pick a single colony, and obtain recombinant bacteria A1-A14 expressing nanobodies.
将重组菌A1-A14接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600达0.6～0.9，取1mL菌液转接至500mL TB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1mM的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎获得纳米抗体粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法纯化纳米抗体。取纯化后的纳米抗体进行SDS-PAGE电泳和Western blot鉴定。从如图25可以看到纳米抗体在大约16kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 Inoculate the recombinant bacteria A1-A14 in 5 mL of LB culture solution containing ampicillin, and culture in a shaker at 37°C until the OD 600 reaches 0.6-0.9, transfer 1 mL of the bacteria liquid to 500 mL of TB culture solution, and shake Cultivate in bed, when the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1mM, culture in a shaker at 28°C for 12-16 hours to induce recombinant bacteria to express the target protein, collect bacterial precipitates by centrifugation, and obtain by ultrasonic crushing The nanobody crude extract was purified by nickel column (purchased from GE Healthcare) affinity chromatography. The purified nanobodies were subjected to SDS-PAGE electrophoresis and Western blot identification. It can be seen from Fig. 25 that the nanobodies have obvious bands at about 16kD, consistent with the expected size of the target fragment, and the purity is over 90%.
2.SPR法鉴定纳米抗体的亲和力2. SPR method to identify the affinity of nanobodies
使用Biacore TM X100蛋白互作仪(购自GE Healthcare公司)鉴定各纳米抗体的与猪CD205目的蛋白的亲和力。首先使用偶联试剂N-ethyl-N’-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide(购自Sigma公司)将1mg实施例13所制备的猪CD205目的蛋白偶联至CM5芯片上，利用生理盐水将纯化后的各纳米抗体(编号如表4所 示)从100nM进行梯度稀释(分别为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM)，并分别与偶联至芯片上的破碎产物进行结合，结合时间180s，使用乙醇胺进行封闭，利用HBS-EP缓冲液(10mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid(HEPES),pH 7.5,150mM NaCl,3.5mM EDTA,and 0.005％(v/v)Surfactant P20)进行洗涤去除未结合的物质，流速为30μL/min，使用10mM glycine/HCl(pH 2.5)进行重生。检测结果经Biacore 2.0.1软件进行分析，结合速率常数(ka)、解离速率常数(kd)以及平衡解离常数(K D)如表12所示。编号为193的纳米抗体K D值达1.04×10 -9，为本发明筛选获得的亲和力最高的纳米抗体，后续研究均围绕该纳米抗体开展。 The affinity of each nanobody to the porcine CD205 target protein was identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare). First, use the coupling reagent N-ethyl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma) to couple 1 mg of the porcine CD205 target protein prepared in Example 13 to the CM5 chip, and use physiological saline The purified Nanobodies (numbered as shown in Table 4) were serially diluted from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and respectively mixed with the fragmented products coupled to the chip For binding, the binding time is 180s, and ethanolamine is used for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v/v) Surfactant P20) to remove unbound material by washing at a flow rate of 30 μL/min and regenerate using 10 mM glycine/HCl (pH 2.5). The test results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 12. The Nanobody No. 193 has a K D value of 1.04×10 -9 , which is the Nanobody with the highest affinity obtained through the screening of the present invention. Subsequent studies will be carried out around this Nanobody.
表12 各纳米抗体与猪CD205目的蛋白结合的动力学参数Table 12 Kinetic parameters of each nanobody binding to porcine CD205 target protein
纳米抗体编号Nanobody ID k a(M -1s -1) k a (M -1 s -1 ) k d(s -1) k d (s -1 ) K D(M) K D (M)
33 2.18×10 4 2.18×10 4 1.71×10 -4 1.71×10 -4 0.78×10 -8 0.78×10 -8
21twenty one 1.29×10 4 1.29×10 4 2.39×10 -4 2.39×10 -4 1.85×10 -8 1.85×10 -8
2525 3.14×10 4 3.14×10 4 1.38×10 -4 1.38×10 -4 0.43×10 -8 0.43×10 -8
4646 2.16×10 4 2.16×10 4 1.26×10 -4 1.26×10 -4 0.58×10 -8 0.58×10 -8
6868 6.91×10 5 6.91×10 5 3.45×10 -4 3.45×10 -4 0.49×10 -9 0.49×10 -9
8787 1.17×10 4 1.17×10 4 3.12×10 -4 3.12×10 -4 2.66×10 -8 2.66×10 -8
8989 4.16×10 4 4.16×10 4 5.10×10 -4 5.10×10 -4 1.23×10 -8 1.23×10 -8
131131 1.47×10 5 1.47×10 5 1.97×10 -4 1.97×10 -4 1.34×10 -9 1.34×10 -9
153153 4.31×10 4 4.31×10 4 3.83×10 -4 3.83×10 -4 8.89×10 -9 8.89×10 -9
169169 2.85×10 4 2.85×10 4 9.52×10 -4 9.52×10 -4 3.34×10 -8 3.34×10 -8
170170 4.25×10 4 4.25×10 4 2.23×10 -4 2.23×10 -4 0.52×10 -8 0.52×10 -8
174174 2.02×10 4 2.02×10 4 1.98×10 -4 1.98×10 -4 0.98×10 -8 0.98×10 -8
181181 2.52×10 4 2.52×10 4 8.65×10 -4 8.65×10 -4 3.43×10 -9 3.43×10 -9
193193 1.99×10 5 1.99×10 5 2.07×10 -4 2.07×10 -4 1.04×10 -9 1.04×10 -9
注：表12中ka代表结合速率常数，kd代表解离速率常数，K D代表平衡解离常数。 Note: In Table 12, ka represents the association rate constant, kd represents the dissociation rate constant, and K D represents the equilibrium dissociation constant.
抽提纳米抗体193的重组菌质粒，送上海生工公司进行序列测定，获得其基因序列和氨基酸序列，其基因序列和氨基酸序列分别如SEQ ID NO.:9和SEQ ID NO.:11所示。The recombinant bacterial plasmid of Nanobody 193 was extracted and sent to Shanghai Shenggong Company for sequence determination to obtain its gene sequence and amino acid sequence. The gene sequence and amino acid sequence are shown in SEQ ID NO.:9 and SEQ ID NO.:11 respectively .
实施例18 针对猪PEDV抗原纳米抗体文库的构建Example 18 Construction of Nanobody Library against Porcine PEDV Antigen
1.RNA的提取与cDNA的合成1. RNA extraction and cDNA synthesis
将1mg猪PEDV抗原(本实验室增殖保存)与弗氏完全佐剂等体积混合，对一只新疆双峰驼进行免疫；1周之后，将1mg猪PEDV抗原与弗氏不完全佐剂等体积混合，免疫该双峰驼，每周一次，共免疫6次，刺激机体产生针对抗原的特异性抗体；免疫结束后，提取100mL骆驼外周血淋巴细胞，提取淋巴细胞的总RNA，按照反转录试剂盒(购自TAKARA公司)说明操作，合成cDNA。Mix 1mg porcine PEDV antigen (proliferated and stored in our laboratory) with Freund's complete adjuvant in equal volume to immunize a Xinjiang Bactrian camel; 1 week later, mix 1mg porcine PEDV antigen with Freund's incomplete adjuvant in equal volume Mix and immunize the Bactrian camel once a week for a total of 6 times to stimulate the body to produce specific antibodies against the antigen; after the immunization, extract 100mL camel peripheral blood lymphocytes, extract the total RNA of the lymphocytes, and follow the reverse transcription The kit (purchased from TAKARA Company) explained the operation and synthesized cDNA.
2.引物的设计与合成2. Design and synthesis of primers
根据参考文献(Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae，Conrath Katja et.al，Antimicrobial Agents and Chemotherapy，2001，45，2807-2812.)设计用于扩增骆驼重链抗体可变区基因VHH片段(350bp)的PCR引物C1F、C1R、V HHF和V HHR。各引物的具体序列如上述表10。 According to references (Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae, Conrath Katja et.al, Antimicrobial Agents and Chemotherapy, 2001, 45, 2807-2812.) designed to amplify camelid heavy chain antibodies can PCR primers C1F, C1R, VHH F and VHH R of the variable region gene VHH fragment (350bp). The specific sequences of each primer are shown in Table 10 above.
3.VHH片段的扩增3. Amplification of VHH fragments
利用本实施例标题1中合成的骆驼cDNA为模板，PCR扩增VHH片段。首先，以cDNA为模板，利用C1F和C1R为上下游引物，扩增获得大小约为750bp的基因片段，反应条件为：95℃3min；95℃30s，59℃1min，72℃1min，共30个循环；72℃10min。反应结束后，回收大小约为750bp的基因片段。然后，以该大小约为750bp的基因片段为模板，利用V HHF和V HHR为上下游引物，扩增VHH片段，反应条件为：95℃3min；95℃30s，58℃1min，72℃30s，共30个循环；72℃10min。反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳进行鉴定，紫外灯下观察目的条带，如图26所示，可见大约350bp的VHH基因片段，与预期大小一致。利用凝胶回收试剂盒(购自TAKARA公司)说明书进行操作，对目的条带进行纯化和回收。 Using the camel cDNA synthesized in Title 1 of this example as a template, the VHH fragment was amplified by PCR. First, using cDNA as a template, using C1F and C1R as upstream and downstream primers, amplified gene fragments with a size of about 750bp, the reaction conditions were: 95°C for 3 minutes; 95°C for 30s, 59°C for 1min, and 72°C for 1min, a total of 30 gene fragments Cycle; 72°C for 10 min. After the reaction, a gene fragment with a size of about 750 bp was recovered. Then, use the gene fragment with a size of about 750bp as a template, and use V HHF and V HHR as upstream and downstream primers to amplify the VHH fragment. The reaction conditions are: 95°C for 3min; 95°C for 30s, 58°C for 1min, and 72°C 30s, 30 cycles in total; 10min at 72°C. After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light, as shown in Figure 26, a VHH gene fragment of about 350bp was seen, which was consistent with the expected size. Purify and recover the target bands by using the instruction manual of the gel recovery kit (purchased from TAKARA company).
4.噬菌体展示基因文库的构建4. Construction of phage display gene library
纯化回收的VHH基因片段采用Pst I和Not I酶切后，连入pMESC载体(购自Novagen公司)。将连接产物转化至E.coli TG1感受态细胞(购自Novagen公司)中，37℃培养1h，将菌液离心浓缩后涂在含有氨苄抗性的LB平板培养基上，37℃过夜生长之后随机挑选24个单克隆菌落，利用V HHF和V HHR为上下游引物进行菌落PCR鉴定。结果如图27，24个单克隆菌落经菌落PCR鉴定，所有单克隆菌落均含有大小约为350bp的目的片段，说明该文库的插入率达到了100％。将上述平板中的菌落刮入LB液体培养基中，然后加入终浓度为30％的甘油，分装保存于-80℃备用，此即为猪PEDV抗原噬菌体展示文库。 The purified and recovered VHH gene fragment was digested with Pst I and Not I, and then ligated into pMESC vector (purchased from Novagen). The ligation product was transformed into E.coli TG1 competent cells (purchased from Novagen), cultured at 37°C for 1 hour, the bacterial liquid was concentrated by centrifugation and spread on the LB plate medium containing ampicillin resistance, after overnight growth at 37°C, randomly 24 single-clonal colonies were selected, and colony PCR identification was carried out using V HHF and V HHR as upstream and downstream primers. The results are shown in Figure 27, 24 monoclonal colonies were identified by colony PCR, and all monoclonal colonies contained the target fragment with a size of about 350bp, indicating that the insertion rate of the library reached 100%. Scrape the bacterium colonies in the above plate into LB liquid medium, then add glycerol with a final concentration of 30%, aliquot and store at -80°C for later use, this is the porcine PEDV antigen phage display library.
实施例19 针对猪PEDV抗原纳米抗体的筛选过程Example 19 Screening process against porcine PEDV antigen nanobody
1.噬菌体展示文库的扩增1. Amplification of phage display library
取200μL于-80℃冻存的实施例18所制备的噬菌体文库，接种至500mL 2×TY培养基中，37℃、摇床转速为200rpm条件下培养3-5h后，加入50μL辅助噬菌体VCSM13(购自Novagen公司)，37℃孵育1h，随后37℃、摇床转速为200rpm条件下培养过夜。次日，加入80g的PEG6000(购自上海生工公司)沉淀噬菌体，该沉淀即为扩增后的噬菌体文库。将扩增后的噬菌体文库重悬于5mL 0.1M PBS中，得到其悬液。Take 200 μL of the phage library prepared in Example 18 that was frozen at -80°C and inoculate it into 500 mL of 2×TY medium. After culturing for 3-5 hours at 37°C with a shaker speed of 200 rpm, add 50 μL of helper phage VCSM13 ( (purchased from Novagen), incubated at 37°C for 1 h, and then cultured overnight at 37°C with a shaker speed of 200 rpm. On the next day, 80 g of PEG6000 (purchased from Shanghai Sangon Co., Ltd.) was added to precipitate the phage, which was the amplified phage library. Resuspend the amplified phage library in 5mL 0.1M PBS to obtain its suspension.
2.亲和筛选2. Affinity screening
将10μg本实验室保藏的猪PEDV抗原加入到10mL、100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入96孔酶标板的每孔中，在4℃包被过夜，设立ST细胞破碎产物(本实验室增殖保存)作为对照；次日，每孔加入100μL、1％脱脂乳溶液，室温封闭2h；然后，每孔加入100μL扩增后的噬菌体文库悬液，室温作用1h，用含有0.05％Tween-20的PBS缓冲液洗5遍，洗掉不结合的噬菌体，随后用100μL、100mM的三乙胺(购自上海生工公司)溶液将与猪PEDV抗原特异性结合的噬菌体洗脱下，并感染5倍体积(约500μL)处于对数期生长(OD 600为0.8)的大肠杆菌TG1细胞，37℃培养1h，加入50μL辅助噬菌体VCSM13(购自Novagen公司)侵染TG1细胞，离心取上清液，即得第一轮筛选到的噬菌体，用于下一轮的筛选。相同筛选过程共进行3轮。每一轮筛选获得的噬菌体各取10μL涂于LB固体培养基上，均在37℃过夜培养，用于观察亲和筛选富集过程。如表13所示，文库经三轮亲和筛选后，每一轮筛选富集到的噬菌体均较上一轮要多。 Add 10 μg of porcine PEDV antigen preserved in our laboratory to 10 mL, 100 mM NaHCO 3 solution (pH 8.2), mix well, take 100 μL and add it to each well of a 96-well microplate plate, coat at 4°C overnight, and set up The disrupted product of ST cells (proliferated and stored in our laboratory) was used as a control; the next day, add 100 μL of 1% skim milk solution to each well, and block at room temperature for 2 hours; then, add 100 μL of amplified phage library suspension to each well, and act for 1 hour at room temperature , washed 5 times with PBS buffer containing 0.05% Tween-20 to wash off unbound phages, and then use 100 μL, 100 mM triethylamine (purchased from Shanghai Shenggong Company) solution to specifically bind porcine PEDV antigen The phage was eluted, and infected with 5 times the volume (about 500 μL) of Escherichia coli TG1 cells in logarithmic growth (OD 600 0.8), cultured at 37°C for 1 hour, and added 50 μL of helper phage VCSM13 (purchased from Novagen) to infect TG1 The cells were centrifuged to take the supernatant, and the phages selected in the first round were obtained for the next round of screening. A total of 3 rounds of the same screening process were performed. 10 μL of the phages obtained in each round of screening were applied to LB solid medium and cultured overnight at 37°C for observing the enrichment process of affinity screening. As shown in Table 13, after three rounds of affinity screening of the library, more phages were enriched in each round of screening than in the previous round.
表13 噬菌体文库3轮亲和筛选富集过程Table 13 Enrichment process of three rounds of affinity screening of phage library
亲和筛选轮次Affinity screening rounds 投入噬菌体文库量(pfu/mL)Amount of phage library input (pfu/mL) 回收噬菌体文库量(pfu/mL)Amount of recovered phage library (pfu/mL)
第一轮亲和筛选First round of affinity screening 1.2×10 7 1.2×10 7 4.1×10 3 4.1×10 3
第二轮亲和筛选Second round of affinity screening 1.1×10 7 1.1×10 7 1.8×10 4 1.8×10 4
第三轮亲和筛选The third round of affinity screening 1.1×10 7 1.1×10 7 2.4×10 5 2.4×10 5
实施例20 酶联免疫方法(ELISA)筛选特异性阳性克隆Example 20 Enzyme-linked immunosorbent assay (ELISA) screening specific positive clones
1.纳米抗体的表达1. Expression of Nanobodies
从实施例19中第三轮筛选后富集在LB平板上的菌落中，挑选200个单菌落分别接种于添加有TB培养基(含有100μg/mL氨苄青霉素)的96孔板中，并设置一个仅加入了TB培养基的空白对照，在37℃、摇床转速为200rpm条件下培养至对数生长期，各孔均加入终浓度为1mM的IPTG，在28℃、摇床转速为200rpm条件下过夜培养。次日，利用超声破碎法分别获得各重组菌表达的纳米抗体，各纳米抗体编号依次为1～200。From the colonies enriched on the LB plate after the third round of screening in Example 19, 200 single colonies were selected and inoculated respectively in 96-well plates supplemented with TB medium (containing 100 μg/mL ampicillin), and a The blank control with only TB medium was cultured to the logarithmic growth phase at 37°C with a shaker speed of 200rpm, and each well was added with a final concentration of 1mM IPTG. Incubate overnight. On the next day, the nanobodies expressed by each recombinant bacteria were respectively obtained by sonicating, and the numbers of each nanobody were 1-200 in sequence.
2.间接ELISA方法检测纳米抗体的结合活性2. Indirect ELISA method to detect the binding activity of nanobodies
采用间接ELISA反应鉴定各纳米抗体与猪PEDV抗原的结合活性。将10μg猪PEDV抗原加入到10mL 100mM的NaHCO 3溶液(pH8.2)中，混合均匀，取100μL加入到96孔酶标板的每孔中，在4℃包被过夜，设立ST细胞破碎产物作为对照；次日，弃掉板内液体，利用含有0.05％Tween-20的PBS缓冲液洗5遍，拍干，每孔加入100μL、5％脱脂乳溶液，室温封闭2h；利用含有0.05％Tween-20的PBS缓冲液洗5遍，将各纳米抗体依次加入ELISA板的各孔中，室温下孵育1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的纳米抗体，加入100μL经1:2000稀释后的Mouse anti-HA tagantibody(鼠抗HA抗体，购自北京康为世纪公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入100μL经1:2000稀释后的HRP labeledgoat anti-mouse IgG(辣根过氧化物酶标记的山羊抗小鼠抗体，购自艾美捷公司)，室温放置1h，利用含有0.05％Tween-20的PBS缓冲液洗去未结合的抗体，加入辣根过氧化物酶显色液(购自上海生工公司)，37℃孵育15min，每孔各加50μL、2M的硫酸溶液终止反应，使用酶标仪测定各孔在450nm波长处的吸光值OD 450。当样品孔OD 450值大于对照孔OD 450值2.5倍以上时，判为阳性克隆孔。结果如图28所示，共有11个纳米抗体可与猪PEDV抗原发生特异性的结合反应(为筛选到高亲和力的纳米抗体，仅选取OD 450值大于2.0的纳米抗体)。 The binding activity of each nanobody to porcine PEDV antigen was identified by indirect ELISA reaction. Add 10 μg of porcine PEDV antigen to 10 mL of 100 mM NaHCO 3 solution (pH 8.2), mix well, take 100 μL and add it to each well of a 96-well microtiter plate, coat at 4°C overnight, and set up the broken product of ST cells as Control; the next day, discard the liquid in the plate, wash 5 times with PBS buffer containing 0.05% Tween-20, pat dry, add 100 μL of 5% skim milk solution to each well, and block at room temperature for 2 hours; Wash 5 times with 20% PBS buffer, add each nanobody to each well of the ELISA plate in turn, incubate at room temperature for 1 h, wash off unbound nanobody with PBS buffer containing 0.05% Tween-20, add 100 μL of :2000 diluted Mouse anti-HA tagantibody (mouse anti-HA antibody, purchased from Beijing Kangwei Century Company), placed at room temperature for 1h, and washed unbound antibody with PBS buffer containing 0.05% Tween-20, added 100 μL of 1:2000 dilution of HRP labeledgoat anti-mouse IgG (horseradish peroxidase-labeled goat anti-mouse antibody, purchased from Amicate), placed at room temperature for 1h, using PBS buffer containing 0.05% Tween-20 Unbound antibodies were washed away, and horseradish peroxidase chromogenic solution (purchased from Shanghai Shenggong Company) was added, incubated at 37°C for 15 min, and 50 μL of 2M sulfuric acid solution was added to each well to terminate the reaction. The absorbance value OD 450 of the hole at a wavelength of 450 nm. When the OD 450 value of the sample well is more than 2.5 times greater than the OD 450 value of the control well, it is judged as a positive clone well. The results are shown in Figure 28. A total of 11 Nanobodies can specifically bind to the porcine PEDV antigen (in order to screen for high-affinity Nanobodies, only Nanobodies with an OD 450 value greater than 2.0 were selected).
3.特异性的鉴定3. Identification of specificity
按照本实施例中标题2所述间接ELISA检测方法，分别检测各纳米抗体与O型FMDV、PCV2、PRRSV、PRV之间的交叉反应性，利用酶标仪测定相应OD 450，不同之处在于ELISA板的包被抗原采用O型FMDV、PCV2、PRRSV、PRV替代PEDV。结果如图29所示，本发明所获得的纳米抗体对O型FMDV、PCV2、PRRSV、PRV交叉反应性极低，说明本发明获得了针对猪PEDV的特异性纳米抗体。 According to the indirect ELISA detection method described in Title 2 of this example, the cross-reactivity between each nanobody and O-type FMDV, PCV2, PRRSV, and PRV was detected respectively, and the corresponding OD 450 was measured by a microplate reader. The difference is that the ELISA The coating antigen of the plate was replaced by PEDV with O-type FMDV, PCV2, PRRSV and PRV. The results are shown in Figure 29, the nanobodies obtained by the present invention have extremely low cross-reactivity to O-type FMDV, PCV2, PRRSV, and PRV, indicating that the present invention has obtained specific nanobodies against porcine PEDV.
实施例21 表面等离子共振法(SPR)筛选高亲和力纳米抗体Example 21 Surface plasmon resonance (SPR) screening of high-affinity nanobodies
1.纳米抗体的表达及纯化1. Expression and purification of nanobodies
分别抽提实施例20所获得的针对猪PEDV抗原的特异性纳米抗体重组菌的质粒，42℃分别转化到大肠杆菌WK6感受态细胞(购自Novagen公司)中，37℃、摇床转速为200rpm的条件下培养1h，离心浓缩菌液，并将其涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16小时；挑选单个菌落，分别获得表达纳米抗体的重组菌B1-B11。The plasmids of the specific nanobody recombinant bacteria directed against porcine PEDV antigen obtained in Example 20 were extracted respectively, and transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C respectively, at 37°C and the shaker speed was 200rpm Cultivate for 1 hour under the condition of 1 h, centrifuge and concentrate the bacterial liquid, spread it on LB plates containing 100 μg/mL ampicillin, and incubate at 37°C for 12 to 16 hours; select a single colony to obtain recombinant bacteria expressing nanobodies B1- B11.
将重组菌B1-B11接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600达0.6～0.9，取1mL菌液转接至500mL TB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1mM的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎获得纳米抗体粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法纯化纳米抗体。取纯化后的纳米抗体 进行SDS-PAGE电泳和Western blot鉴定。从如图30可以看到纳米抗体在大约16kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 Inoculate the recombinant bacteria B1-B11 in 5 mL of LB culture solution containing ampicillin, and cultivate in a shaker at 37°C until the OD 600 reaches 0.6-0.9, transfer 1 mL of the bacteria liquid to 500 mL of TB culture solution, and shake Cultivate in bed, when the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1mM, culture in a shaker at 28°C for 12-16 hours to induce recombinant bacteria to express the target protein, collect bacterial precipitates by centrifugation, and obtain by ultrasonic crushing The nanobody crude extract was purified by nickel column (purchased from GE Healthcare) affinity chromatography. The purified nanobodies were subjected to SDS-PAGE electrophoresis and Western blot identification. From Figure 30, it can be seen that the Nanobodies have obvious bands at about 16kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
2.SPR法鉴定纳米抗体的亲和力2. SPR method to identify the affinity of nanobodies
使用Biacore TM X100蛋白互作仪(购自GE Healthcare公司)鉴定各纳米抗体与猪PEDV抗原的亲和力。首先使用偶联试剂N-ethyl-N’-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide(购自Sigma公司)将1mg猪PEDV抗原偶联至CM5芯片上，利用生理盐水将纯化后的各纳米抗体(编号如表6所示)从100nM进行梯度稀释(分别为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM)，并分别与偶联至芯片上的破碎产物进行结合，结合时间180s，使用乙醇胺进行封闭，利用HBS-EP缓冲液(10mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid(HEPES),pH 7.5,150mM NaCl,3.5mM EDTA,and 0.005％(v/v)Surfactant P20)进行洗涤去除未结合的物质，流速为30μL/min，使用10mM glycine/HCl(pH 2.5)进行重生。检测结果经Biacore 2.0.1软件进行分析，结合速率常数(ka)、解离速率常数(kd)以及平衡解离常数(K D)如表14所示。编号为2的纳米抗体K D值达1.03×10 -8，为本发明筛选获得的亲和力最高的纳米抗体，后续研究均围绕该纳米抗体开展。 The affinity of each Nanobody to the porcine PEDV antigen was identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare). First, use the coupling reagent N-ethyl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma) to couple 1 mg of porcine PEDV antigen to the CM5 chip, and use physiological saline to mix the purified nanobodies (The numbers are shown in Table 6) from 100nM to carry out serial dilution (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and respectively combined with the broken product coupled to the chip, the binding time is 180s, Use ethanolamine to block, utilize HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v/v ) Surfactant P20) for washing to remove unbound substances at a flow rate of 30 μL/min and regenerated using 10 mM glycine/HCl (pH 2.5). The test results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 14. The Nanobody No. 2 has a K D value of 1.03×10 -8 , which is the Nanobody with the highest affinity obtained through the screening of the present invention. Subsequent studies will be carried out around this Nanobody.
表14 各纳米抗体与猪PEDV抗原结合的动力学参数Table 14 Kinetic parameters of each Nanobody binding to porcine PEDV antigen
纳米抗体编号Nanobody ID k a(M -1s -1) k a (M -1 s -1 ) k d(s -1) k d (s -1 ) K D(M) K D (M)
22 1.15×10 4 1.15×10 4 1.19×10 -4 1.19×10 -4 1.03×10 -8 1.03×10 -8
1616 1.25×10 4 1.25×10 4 2.31×10 -4 2.31×10 -4 1.84×10 -8 1.84×10 -8
2525 2.36×10 4 2.36×10 4 4.79×10 -4 4.79×10 -4 2.02×10 -8 2.02×10 -8
3737 3.73×10 4 3.73×10 4 2.85×10 -4 2.85×10 -4 0.76×10 -8 0.76×10 -8
6161 1.82×10 4 1.82×10 4 2.19×10 -4 2.19×10 -4 1.2×10 -8 1.2×10 -8
6767 3.42×10 4 3.42×10 4 1.69×10 -4 1.69×10 -4 0.49×10 -8 0.49×10 -8
8888 1.85×10 4 1.85×10 4 5.79×10 -4 5.79×10 -4 3.12×10 -8 3.12×10 -8
122122 4.39×10 4 4.39×10 4 3.29×10 -4 3.29×10 -4 0.74×10 -8 0.74×10 -8
149149 5.46×10 4 5.46×10 4 1.72×10 -4 1.72×10 -4 0.31×10 -8 0.31×10 -8
170170 3.71×10 4 3.71×10 4 4.99×10 -4 4.99×10 -4 1.34×10 -8 1.34×10 -8
191191 5.28×10 4 5.28×10 4 7.11×10 -4 7.11×10 -4 1.35×10 -8 1.35×10 -8
注：表6中ka代表结合速率常数，kd代表解离速率常数，K D代表平衡解离常数。 Note: In Table 6, ka represents the association rate constant, kd represents the dissociation rate constant, and K D represents the equilibrium dissociation constant.
抽提纳米抗体2的重组菌质粒，送上海生工公司进行序列测定，获得其基因序列和氨基酸序列，其基因序列和氨基酸序列分别如SEQ ID NO.:13和SEQ ID NO.:14所示。Extract the recombinant bacterial plasmid of Nanobody 2 and send it to Shanghai Shenggong Company for sequence determination to obtain its gene sequence and amino acid sequence, which are shown in SEQ ID NO.:13 and SEQ ID NO.:14 respectively .
实施例22 双功能纳米抗体Nb193-2的串联表达及纯化Example 22 Tandem expression and purification of bifunctional nanobody Nb193-2
1.双功能纳米抗体Nb193-2基因片段的构建1. Construction of bifunctional nanobody Nb193-2 gene fragment
分别抽提表达猪CD205特异性纳米抗体193和表达猪PEDV抗原特异性纳米抗体2的重组菌质粒，分别利用表15所示的引物NbF、NbLR和NbLF、NbR进行扩增，获得猪CD205特异性的纳米抗体193和猪PEDV抗原特异性的纳米抗体2的基因片段，并进行纯化回收。利用纯化回收的纳米抗体基因片段为模板，以(G4S)4序列为接头元件linker，通过重叠延伸拼接(Splicing by Overlap Extension,SOE)PCR方法构建双功能纳米抗体片段Nb193-2，反应分两步进行：The plasmids of recombinant bacteria expressing porcine CD205-specific nanobody 193 and porcine PEDV antigen-specific nanobody 2 were extracted respectively, and amplified using the primers NbF, NbLR, NbLF, and NbR shown in Table 15 to obtain porcine CD205-specific Nanobody 193 and porcine PEDV antigen-specific nanobody 2 gene fragments were purified and recovered. Using the purified and recovered nanobody gene fragment as a template and (G4S)4 sequence as a linker element linker, the bifunctional nanobody fragment Nb193-2 was constructed by splicing by Overlap Extension (SOE) PCR method, and the reaction was divided into two steps conduct:
第一步反应是在不加引物的条件下进行，反应条件为：95℃3min；95℃30s，62℃30s，72℃2min，共8个循环；72℃10min；The first step reaction is carried out without primers, and the reaction conditions are: 95°C for 3min; 95°C for 30s, 62°C for 30s, 72°C for 2min, a total of 8 cycles; 72°C for 10min;
第二步反应只需要在原始PCR管内加入引物NbF和NbR即可，反应条件为：95℃ 3min；95℃30s，55℃30s，72℃2min，共25个循环；72℃10min；The second step of the reaction only needs to add primers NbF and NbR to the original PCR tube. The reaction conditions are: 95°C for 3min; 95°C for 30s, 55°C for 30s, 72°C for 2min, a total of 25 cycles; 72°C for 10min;
反应结束后，PCR扩增产物经1％琼脂糖凝胶电泳鉴定，紫外灯下观察目的条带，如图31所示，可见大约900bp的基因片段，与预期片段大小相一致，随后插入pMECS载体(购自Novagen公司)的Pst I和Xba I酶切位点之间，于42℃转化到大肠杆菌WK6感受态细胞(购自Novagen公司)，37℃、摇床转速为200rpm的条件下培养1h，离心浓缩菌液，并将其涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃培养12～16小时；挑选单个菌落，得到表达双功能纳米抗体的重组菌C。各引物的具体序列如表15：After the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the target band was observed under ultraviolet light. As shown in Figure 31, a gene fragment of about 900bp was seen, which was consistent with the expected fragment size, and then inserted into the pMECS vector (purchased from Novagen) between the Pst I and Xba I restriction sites, transformed into Escherichia coli WK6 competent cells (purchased from Novagen) at 42°C, and cultured for 1h at 37°C with a shaker speed of 200rpm , concentrate the bacterial solution by centrifugation, spread it on an LB plate containing 100 μg/mL ampicillin, and incubate at 37° C. for 12 to 16 hours; select a single colony to obtain recombinant bacteria C expressing bifunctional nanobodies. The specific sequence of each primer is shown in Table 15:
表15 SOE-PCR扩增引物Table 15 SOE-PCR amplification primers
Figure PCTCN2021126429-appb-000004
Figure PCTCN2021126429-appb-000004
2.双功能纳米抗体基因序列的鉴定2. Identification of bifunctional nanobody gene sequence
抽提表达双功能纳米抗体Nb193-2重组菌C的质粒，送上海生工公司进行序列测定，获得接头元件(G4S)4和双功能纳米抗体Nb193-2的基因序列以及氨基酸序列。接头元件(G4S)4和双功能纳米抗体Nb193-2的基因序列分别如SEQ ID NO.:3和SEQ ID NO.:15所示，接头元件(G4S)4和双功能纳米抗体Nb193-2的氨基酸序列分别如SEQ ID NO.:7和SEQ ID NO.:16所示。The plasmid expressing the recombinant bacteria C of the bifunctional nanobody Nb193-2 was extracted and sent to Shanghai Sangong Company for sequence determination to obtain the gene sequence and amino acid sequence of the linker element (G4S) 4 and the bifunctional nanobody Nb193-2. The gene sequences of linker element (G4S) 4 and bifunctional Nanobody Nb193-2 are shown in SEQ ID NO.: 3 and SEQ ID NO.: 15 respectively, and the gene sequences of linker element (G4S) 4 and bifunctional Nanobody Nb193-2 The amino acid sequences are respectively shown in SEQ ID NO.:7 and SEQ ID NO.:16.
3.双功能纳米抗体Nb193-2的表达与纯化鉴定3. Expression, purification and identification of bifunctional nanobody Nb193-2
利用重组菌制备双功能纳米抗体Nb193-2。具体方法如下：将重组菌C接种在5mL含有氨苄青霉素的LB培养液中，在37℃摇床中培养至OD 600＝0.6～0.9，取1mL菌液转接至500mL TB培养液中，在37℃摇床中培养，当OD 600值达到0.6～0.9时，加入终浓度为1mM的IPTG，在28℃摇床中培养12～16小时诱导重组菌表达目的蛋白，离心收集菌体沉淀，利用超声破碎菌体，取裂解液作为双功能纳米抗体粗提液，采用镍柱(购自GE Healthcare公司)亲和层析法进行纯化。取纯化后的双功能纳米抗体Nb193-2进行SDS-PAGE电泳和Western blot鉴定。从图32可以看到双功能纳米抗体Nb193-2在大约35kD处均有明显条带，与目的片段预期大小相一致，纯度达90％以上。 The bifunctional nanobody Nb193-2 was prepared by recombinant bacteria. The specific method is as follows: inoculate the recombinant bacteria C in 5 mL of LB culture solution containing ampicillin, and cultivate it in a shaker at 37°C until OD 600 =0.6-0.9, transfer 1 mL of the bacteria solution to 500 mL of TB culture solution, and incubate at 37°C. Cultivate in a shaker at ℃, when the OD 600 value reaches 0.6-0.9, add IPTG with a final concentration of 1mM, culture in a shaker at 28°C for 12-16 hours to induce recombinant bacteria to express the target protein, collect the bacterial precipitate by centrifugation, and use ultrasonic The bacterium was broken, and the lysate was taken as the crude extract of the bifunctional nanobody, which was purified by affinity chromatography using a nickel column (purchased from GE Healthcare). The purified bifunctional nanobody Nb193-2 was identified by SDS-PAGE electrophoresis and Western blot. From Figure 32, it can be seen that the bifunctional nanobody Nb193-2 has an obvious band at about 35kD, which is consistent with the expected size of the target fragment, and the purity is over 90%.
实施例23 表面等离子共振法(SPR)鉴定双功能纳米抗体Nb193-2的亲和力Example 23 Surface plasmon resonance (SPR) identification of the affinity of the bifunctional nanobody Nb193-2
1.SPR法鉴定纳米抗体的亲和力1. SPR method to identify the affinity of nanobodies
使用Biacore TM X100蛋白互作仪(购自GE Healthcare公司)分别鉴定双功能纳米抗体193-2与猪CD205目的蛋白以及猪PEDV抗原的亲和力。首先使用偶联试剂N-eth yl-N’-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide(购自Sigma公司)分别将1mg猪CD205目的蛋白和1mg猪PEDV抗原偶联至CM5芯片上，利用生理盐水将纯化后的双功能纳米抗体从100nM进行梯度稀释(分别为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM)，并分别与偶联至芯片上的破碎产物进行结合，结合时间180s，使用乙醇胺进行封闭，利用HBS-EP缓冲液(10mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid(HEPES),pH 7.5,150mM NaCl,3.5mM EDTA,and 0.005％(v/v)Surfactant P20)进行洗涤去除未结合的物质，流速为30μL/min，使用10mM glycine/HCl(pH 2.5)进行重生。检测结果经Biacore 2.0.1软件进行分析，结合速率常数(ka)、解离速率常数(kd)以及平衡解离常数(K D)如表8所示。双功能纳米抗 体Nb193-2与猪CD205目的蛋白、猪PEDV抗原的平衡解离常数(K D)分别为1.52×10 -8、1.01×10 -8。 The affinities of the bifunctional nanobody 193-2 to the porcine CD205 target protein and the porcine PEDV antigen were respectively identified using a Biacore TM X100 protein interaction instrument (purchased from GE Healthcare). First, use the coupling reagent N-eth yl-N'-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide (purchased from Sigma) to couple 1 mg of porcine CD205 target protein and 1 mg of porcine PEDV antigen to the CM5 chip respectively, and use physiological Saline diluted the purified bifunctional nanobody from 100nM (respectively 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and combined with the broken products coupled to the chip respectively, the binding time was 180s , using ethanolamine for blocking, using HBS-EP buffer (10mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 150mM NaCl, 3.5mM EDTA, and 0.005% (v/ v) Surfactant P20) was washed to remove unbound material at a flow rate of 30 μL/min and regenerated with 10 mM glycine/HCl (pH 2.5). The detection results were analyzed by Biacore 2.0.1 software, and the association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K D ) are shown in Table 8. The equilibrium dissociation constants (K D ) of bifunctional nanobody Nb193-2 with porcine CD205 target protein and porcine PEDV antigen are 1.52×10 -8 and 1.01×10 -8 , respectively.
表16.双功能纳米抗体193-2与抗原结合的动力学参数Table 16. Kinetic parameters of binding of bifunctional Nanobody 193-2 to antigen
抗原antigen k a(M -1s -1) k a (M -1 s -1 ) k d(s -1) k d (s -1 ) K D(M) K D (M)
猪CD205目的蛋白Pig CD205 target protein 3.41×10 4 3.41×10 4 5.21×10 -4 5.21×10 -4 1.52×10 -8 1.52×10 -8
猪PEDV抗原porcine PEDV antigen 1.03×10 4 1.03×10 4 1.04×10 -4 1.04×10 -4 1.01×10 -8 1.01×10 -8
注：表16中ka代表结合速率常数，kd代表解离速率常数，K D代表平衡解离常数。 Note: In Table 16, ka represents the association rate constant, kd represents the dissociation rate constant, and K D represents the equilibrium dissociation constant.
实施例24 激光共聚焦显微镜技术鉴定双功能纳米抗体Nb193-2的抗原提呈作用Example 24 Identification of Antigen Presentation of Bifunctional Nanobody Nb193-2 by Confocal Laser Microscopy
1.猪BMDC细胞的诱导分化1. Induced differentiation of porcine BMDC cells
收集新鲜的猪骨髓祖细胞(4×10 6cells/mL)，使用猪重组GM-CSF(20ng/mL，购自上海优宁维公司)和猪重组IL-4(10ng/mL，购自上海优宁维公司)对其进行诱导分化，连续培养7天后，收集猪BMDC细胞并种植于细胞培养皿中(2×10 6cells/mL)，并用4％多聚甲醛室温固定10min，PBS清洗3遍后置于含0.1％Triton的PBS中37℃处理5min，用PBS清洗3遍后置于封闭液中37℃封闭1h，用PE标记anti-porcine CD11c荧光抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤细胞三遍，使用FITC标记anti-porcine CD11b荧光抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤细胞三遍，使用DAPI(工作液，购自上海碧云天公司)染色10min，PBS洗涤三遍后置于激光共聚焦显微镜下进行观察，如图33所示，获得了较为丰富的猪BMDC细胞。 Collect fresh porcine bone marrow progenitor cells (4×10 6 cells/mL), use porcine recombinant GM-CSF (20ng/mL, purchased from Shanghai Unionwell Company) and porcine recombinant IL-4 (10ng/mL, purchased from Shanghai Unionville Company) to induce differentiation, and after continuous culture for 7 days, porcine BMDC cells were collected and planted in cell culture dishes (2×10 6 cells/mL), fixed with 4% paraformaldehyde at room temperature for 10 min, and washed with PBS for 3 After one pass, they were treated in PBS containing 0.1% Triton at 37°C for 5 minutes, washed three times with PBS, and blocked in blocking solution for 1 hour at 37°C, and PE-labeled anti-porcine CD11c fluorescent antibody (diluted at 1:1000, purchased from Shanghai Unionville Company) was incubated at 4°C for 30 min, cells were washed three times with PBS, and FITC-labeled anti-porcine CD11b fluorescent antibody (diluted at 1:1000, purchased from Shanghai Unionwell Company) was incubated at 4°C for 30 min, cells were washed three times with PBS, DAPI (working solution, purchased from Shanghai Biyuntian Company) was used to stain for 10 minutes, washed three times with PBS, and observed under a laser confocal microscope. As shown in FIG. 33 , relatively abundant porcine BMDC cells were obtained.
2.激光共聚焦显微镜技术鉴定双功能纳米抗体Nb193-2的PEDV抗原递送能力2. Identification of PEDV antigen delivery ability of bifunctional nanobody Nb193-2 by confocal laser microscopy
将实施例22所制备的双功能纳米抗体Nb193-2进行柱层析去除内毒素，并使用内毒素检测试剂盒(购自Pyrosate公司，0.25EU/mL)检测其内毒素含量，确保内毒素水平小于0.05EU，并使用FITC荧光染料(购自上海优宁维公司)对其进行标记，超滤(4000g，20min)除去未标记上的多余染料。运用蔗糖密度梯度离心技术对猪PEDV抗原进行纯化，并利用SDS-PAGE和Western Blotting方法对纯化产物进行鉴定。按照本实施例标题1所述方法诱导分化新鲜的猪BMDC细胞，使用FITC荧光染料标记的双功能纳米抗体(5μg/mL)与纯化后的PEDV抗原(5μg/mL)4℃孵育30min后，再与猪BMDC细胞(1×10 6cells/mL)4℃孵育30min，收集细胞，并用4％多聚甲醛室温固定10min，PBS清洗三遍后置于含0.1％Triton的PBS中37℃处理5min，用PBS清洗三遍后置于封闭液中37℃封闭1h，PBS洗涤三遍，使用AF647标记的抗猪CD1抗体(1：1000稀释，购自上海优宁维公司)4℃孵育30min，PBS洗涤三遍，使用DAPI(工作液，购自上海碧云天公司)染色10min，PBS洗涤三遍后置于激光共聚焦显微镜下进行观察。结果如图34所示，加入双功能纳米抗体193-2的猪BMDC细胞试验组绿色荧光强度高于对照组，表明PEDV抗原经过双功能纳米抗体193-2递送至猪BMDC细胞。 The bifunctional nanobody Nb193-2 prepared in Example 22 was subjected to column chromatography to remove endotoxin, and the endotoxin content was detected using an endotoxin detection kit (purchased from Pyrosate Company, 0.25EU/mL) to ensure the endotoxin level It is less than 0.05 EU, and it is labeled with FITC fluorescent dye (purchased from Shanghai Unionwell Co., Ltd.), and the unlabeled excess dye is removed by ultrafiltration (4000g, 20min). The porcine PEDV antigen was purified by sucrose density gradient centrifugation, and the purified product was identified by SDS-PAGE and Western Blotting. According to the method described in Title 1 of this example, fresh porcine BMDC cells were induced to differentiate, and FITC fluorescent dye-labeled bifunctional nanobody (5 μg/mL) was used to incubate with purified PEDV antigen (5 μg/mL) for 30 minutes at 4° C., and then Incubate with porcine BMDC cells (1×10 6 cells/mL) at 4°C for 30 min, collect the cells, fix with 4% paraformaldehyde at room temperature for 10 min, wash with PBS three times, and place in PBS containing 0.1% Triton for 5 min at 37°C. After washing with PBS for three times, place in blocking solution for 1 h at 37°C, wash with PBS three times, use AF647-labeled anti-pig CD1 antibody (diluted 1:1000, purchased from Shanghai Univ Company) to incubate at 4°C for 30 min, wash with PBS Three times, stained with DAPI (working solution, purchased from Shanghai Biyuntian Company) for 10 min, washed three times with PBS, and placed under a laser confocal microscope for observation. The results are shown in Figure 34. The green fluorescence intensity of the pig BMDC cell test group added with the bifunctional nanobody 193-2 was higher than that of the control group, indicating that the PEDV antigen was delivered to the pig BMDC cells through the bifunctional nanobody 193-2.
实施例25 双功能纳米抗体Nb193-2提呈PEDV抗原的免疫效力评价Example 25 Evaluation of the immune efficacy of PEDV antigen presented by bifunctional nanobody Nb193-2
1.免疫试验1. Immunization test
将实施例22所制备的双功能纳米抗体Nb193-2(5μg/mL)与纯化后的PEDV抗原(5μg/mL)4℃孵育60min后，辅以206免疫佐剂，对猪进行免疫实验，每只猪颈部肌肉免疫2mL，并于28d进行采血取样，同时设置双功能纳米抗体配伍PBS对照组、非CD205靶向纳米抗体配伍PEDV对照组、非CD205靶向纳米抗体配伍PBS对照组、PEDV对照组和PBS空白对照组，每组5只猪。After incubating the bifunctional nanobody Nb193-2 (5 μg/mL) prepared in Example 22 and the purified PEDV antigen (5 μg/mL) at 4°C for 60 min, supplemented with 206 immune adjuvant, the pig was immunized with an experiment. Pig neck muscles were immunized with 2 mL, and blood samples were collected on the 28th day. At the same time, a bifunctional nanobody-compatible PBS control group, a non-CD205-targeting nanobody-compatible PEDV control group, a non-CD205-targeting nanobody-compatible PBS control group, and a PEDV control group were set. group and PBS blank control group, with 5 pigs in each group.
2.免疫效力评价2. Evaluation of immune efficacy
利用PEDV抗体检测试剂盒(购自武汉科前公司)检测免后血清中特异性抗体水平、 抗体亚型(IgG1和IgG2a)及IgA抗体水平，检测步骤均按照试剂盒说明书进行操作。抗体检测结果如图35所示，双功能纳米抗体配伍PEDV试验组的免后28天抗体效价明显高于其他试验组和对照组，表明双功能纳米抗体对PEDV抗原免后抗体产生具有促进作用；抗体亚型检测结果表明(如图36，37所示)，双功能纳米抗体配伍PEDV试验组的免后28天IgG1和IgG2a抗体效价均明显高于其他试验组和对照组，表明双功能纳米抗体可以显著提升PEDV抗原免后IgG1和IgG2a抗体水平；IgA检测结果显示(如图38所示)，尽管各试验组和对照组的抗体水平较低，但双功能纳米抗体配伍PEDV试验组的免后28天粘膜IgA抗体效价明显高于其他试验组和对照组，表明双功能纳米抗体可以显著提升PEDV抗原免后粘膜IgA抗体水平。The PEDV antibody detection kit (purchased from Wuhan Keqian Company) was used to detect the specific antibody level, antibody subtype (IgG1 and IgG2a) and IgA antibody level in the serum after immunization, and the detection steps were all performed according to the kit instructions. The results of antibody detection are shown in Figure 35. The antibody titer of the bifunctional nanobody-compatible PEDV test group 28 days after immunization was significantly higher than that of the other test groups and the control group, indicating that the bifunctional nanobody can promote the production of antibodies to PEDV antigens after immunization ; Antibody subtype detection results showed (as shown in Figure 36, 37), the IgG1 and IgG2a antibody titers of the bifunctional nanobody compatibility PEDV test group were significantly higher than other test groups and control groups 28 days after immunization, indicating that the bifunctional nanobody Nanobodies can significantly increase the levels of IgG1 and IgG2a antibodies after PEDV antigen immunization; IgA test results show (as shown in Figure 38), although the antibody levels of each test group and control group are low, the bifunctional nanobody compatibility PEDV test group The titer of mucosal IgA antibody 28 days after immunization was significantly higher than that of other test groups and control groups, indicating that the bifunctional nanobody can significantly increase the level of mucosal IgA antibody after immunization with PEDV antigen.
于注射位点附近活体采集免疫后猪的新鲜淋巴结，制备淋巴结单细胞悬液，向细胞悬液中加入淋巴细胞分离液，进行密度梯度离心(3000g，20min)，吸取淋巴细胞(离心后从上到下一共分为四层，吸取第二层)，用含血清的细胞培养基洗涤3次后接种至96孔板中(1×10 6cells/mL)；分别用纯化后的PEDV抗原(5μg/mL)、LPS刺激细胞，置于细胞培养箱中(37℃，含5％CO 2)继续培养48h；每孔分别加入MTT(5mg/mL)后继续培养4h，离心收集细胞样品，加入DMSO并混匀至晶体溶解，将细胞板置于酶标仪中读取结果。结果如图39所示，与对照组相比，Nb193-2+PEDV试验组可以显著提高淋巴细胞的增殖水平，诱导更好的细胞免疫水平。 Fresh lymph nodes of immunized pigs were collected near the injection site, and lymph node single cell suspension was prepared. Lymphocyte separation medium was added to the cell suspension, and density gradient centrifugation (3000g, 20min) was carried out. Divided into four layers in total, aspirate the second layer), washed 3 times with serum-containing cell culture medium and inoculated into 96-well plates (1×10 6 cells/mL); purified PEDV antigen (5 μg /mL), LPS stimulated cells, placed in a cell culture incubator (37°C, containing 5% CO 2 ) and continued to culture for 48h; MTT (5mg/mL) was added to each well and cultured for 4h, cell samples were collected by centrifugation, and DMSO was added And mix until the crystals are dissolved, and place the cell plate in a microplate reader to read the results. The results are shown in Figure 39. Compared with the control group, the Nb193-2+PEDV test group can significantly increase the proliferation level of lymphocytes and induce a better level of cellular immunity.
将上述新鲜制备的淋巴细胞(2×10 6cells/mL)接种至24孔板中，用纯化后的PEDV抗原(5μg/mL)体外刺激淋巴细胞，置于细胞培养箱中(37℃，含5％CO 2)共同作用120min，收集部分细胞样品并离心取上清液，按照ELISA试剂盒(购自上海优宁维公司)说明对上清液中IFN-γ、IL-6、IL-4的浓度进行检测，结果如图40所示，Nb193-2+PEDV试验组IFN-γ、IL-6、IL-4分泌水平均显著高于其余试验组和对照组，进一步说明双功能纳米抗体可以显著提高PEDV抗原免疫后所引起的细胞免疫反应。 Inoculate the above-mentioned freshly prepared lymphocytes (2×10 6 cells/mL) into 24-well plates, stimulate the lymphocytes in vitro with purified PEDV antigen (5 μg/mL), and place them in a cell culture incubator (37°C, containing 5% CO 2 ) for 120 min, some cell samples were collected and centrifuged to get the supernatant, and the IFN-γ, IL-6, IL-4 As shown in Figure 40, the secretion levels of IFN-γ, IL-6, and IL-4 in the Nb193-2+PEDV test group were significantly higher than those of the rest of the test group and the control group, further illustrating that bifunctional nanobodies can Significantly improve the cellular immune response caused by PEDV antigen immunization.
本发明是结合最佳实施例进行描述的，然而在阅读了本发明的上述内容后，本领域技术人员会容易意识到，本发明可容易改造而获得本文所述的那些目的和优点以及隐含在本文中的那些目的和优点。在本文中以当前优选实施方式的代表形式描述的方法、变体和组合物是示例性的，并不意在限制本发明的范围。对于本领域技术人员来说，可对它们做出改变或将其用于其他用途，但这都包括在本申请所附权利要求书定义的本发明的范围内。The present invention is described in conjunction with the best embodiment, but after reading the above content of the present invention, those skilled in the art will easily realize that the present invention can be easily modified to obtain those objects and advantages described herein and the implicit Objects and advantages of those in this paper. The methods, variants and compositions described herein as representative of presently preferred embodiments are exemplary and are not intended to limit the scope of the invention. It is possible for those skilled in the art to make changes to them or to use them for other purposes, but this is included within the scope of the present invention as defined by the appended claims of the present application.

Claims (26)

  1. 基于DC细胞的双功能纳米抗体，其特征在于，所述双功能纳米抗体是以接头元件将基于DC细胞的目的蛋白特异性纳米抗体的基因和编码粒径为150nm以下的病毒抗原特异性纳米抗体的基因连接后获得目的基因片段并重组表达获得的目的蛋白。The bifunctional nanobody based on DC cells is characterized in that, the bifunctional nanobody uses a linker element to combine the gene of the target protein-specific nanobody based on DC cells and the virus antigen-specific nanobody whose encoding particle size is below 150nm The target gene fragments are obtained after the genes are connected, and the obtained target protein is recombinantly expressed.
  2. 根据权利要求1所述的基于DC细胞的双功能纳米抗体，其特征在于，所述基于DC细胞的目的蛋白特异性纳米抗体包括编码DC细胞特异性的纳米抗体或编码DC细胞抗原提呈受体特异性的纳米抗体。The DC cell-based bifunctional nanobody according to claim 1, wherein the DC cell-based target protein-specific nanobody comprises a DC cell-specific nanobody or a DC cell antigen-presenting receptor specific nanobodies.
  3. 根据权利要求1所述的基于DC细胞的双功能纳米抗体，其特征在于，所述病毒抗原包括猪圆环病毒抗原、猪细小病毒抗原、猪口蹄疫病毒抗原、猪瘟病毒抗原、猪繁殖与呼吸综合征病毒抗原或猪流行性腹泻病毒抗原中的一种或几种。The DC cell-based bifunctional nanobody according to claim 1, wherein the viral antigens include porcine circovirus antigens, porcine parvovirus antigens, porcine foot-and-mouth disease virus antigens, swine fever virus antigens, porcine reproductive and respiratory One or more of syndrome virus antigens or porcine epidemic diarrhea virus antigens.
  4. 权利要求1所述的基于DC细胞的双功能纳米抗体的制备方法，其特征在于，包括以下步骤：The method for preparing a DC cell-based bifunctional nanobody according to claim 1, comprising the following steps:
    1)使用新鲜诱导分化的DC细胞免疫骆驼或羊驼，经过多次免疫后，提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库；1) Using freshly differentiated DC cells to immunize camels or alpacas, after multiple immunizations, extract the cDNA of peripheral blood lymphocytes, and amplify the antibody heavy chain variable region gene library to construct a phage display nanobody gene library;
    2)将新鲜诱导分化的DC细胞作为抗原进行包被，进行3-5轮亲和筛选，获得DC特异性纳米抗体；2) Coating freshly induced differentiated DC cells as antigens, and performing 3-5 rounds of affinity screening to obtain DC-specific nanobodies;
    3)使用粒径为150nm以下的病毒免疫骆驼或羊驼，经过多次免疫后，提取其外周血淋巴细胞cDNA，并扩增其抗体重链可变区基因文库，构建噬菌体展示纳米抗体基因文库；3) Use viruses with a particle size of 150nm or less to immunize camels or alpacas. After multiple immunizations, extract the cDNA of peripheral blood lymphocytes and amplify the antibody heavy chain variable region gene library to construct a phage display nanobody gene library ;
    4)将粒径为150nm以下的病毒作为抗原进行包被，进行3-5轮亲和筛选，获得所述的病毒特异性纳米抗体；4) Coating a virus with a particle size below 150nm as an antigen, and performing 3-5 rounds of affinity screening to obtain the virus-specific nanobody;
    5)将DC特异性纳米抗体的基因序列和所述的病毒病原特异性纳米抗体的基因序列连接后获得目的基因片段并重组表达获得的目的蛋白。5) After linking the gene sequence of the DC-specific Nanobody and the gene sequence of the virus pathogen-specific Nanobody, the target gene fragment is obtained and the obtained target protein is recombinantly expressed.
  5. 靶向猪DC细胞的猪O型FMDV的双功能纳米抗体，其特征在于，所述靶向猪DC细胞的猪O型FMDV的双功能纳米抗体是以接头元件将编码猪DC特异性的纳米抗体Nb131的基因和编码猪O型FMDV特异性的纳米抗体Nb104的基因连接后获得目的基因片段进一步重组表达获得的目的蛋白。The bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells is characterized in that the bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells uses a linker element to encode a porcine DC-specific nanobody The gene of Nb131 and the gene encoding pig O-type FMDV-specific Nanobody Nb104 are connected to obtain the target gene fragment and further recombine and express the target protein obtained.
  6. 根据权利要求5所述的靶向猪DC细胞的猪O型FMDV的双功能纳米抗体，其特征在于，所述猪DC特异性的纳米抗体Nb131的氨基酸序列为SEQ ID NO.:5所示，所述猪O型FMDV特异性的纳米抗体Nb104的氨基酸序列为SEQ ID NO.:6所示。The bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells according to claim 5, wherein the amino acid sequence of the porcine DC-specific nanobody Nb131 is shown in SEQ ID NO.:5, The amino acid sequence of the pig O-type FMDV-specific Nanobody Nb104 is shown in SEQ ID NO.:6.
  7. 根据权利要求5所述的靶向猪DC细胞的猪O型FMDV的双功能纳米抗体，其特征在于，所述编码猪DC特异性的纳米抗体Nb131的基因的核苷酸序列为SEQ ID NO.:1所示，所述编码猪O型FMDV特异性的纳米抗体Nb104的基因的核苷酸序列为SEQ ID NO.:2所示。The bifunctional nanobody of the porcine O-type FMDV targeting porcine DC cells according to claim 5, wherein the nucleotide sequence of the gene of the porcine DC-specific nanobody Nb131 is SEQ ID NO. Shown in: 1, the nucleotide sequence of the gene of the nanobody Nb104 of described coding porcine O type FMDV specificity is shown in SEQ ID NO.:2.
  8. 根据权利要求5所述的靶向猪DC细胞的猪O型FMDV双功能纳米抗体，其特征在于，所述接头元件为接头元件(G4S)4，所述接头元件(G4S)4的核苷酸序列如SEQ ID NO.:3所示，接头元件(G4S)4的氨基酸序列如SEQ ID NO.:7所示。The porcine O-type FMDV bifunctional nanobody targeting porcine DC cells according to claim 5, wherein the linker element is a linker element (G4S) 4, and the nucleotide of the linker element (G4S) 4 The sequence is shown in SEQ ID NO.: 3, and the amino acid sequence of the linker element (G4S) 4 is shown in SEQ ID NO.: 7.
  9. 根据权利要求5所述的靶向猪DC细胞的猪O型FMDV双功能纳米抗体，其特征在于，所述靶向猪DC细胞的猪O型FMDV双功能纳米抗体的氨基酸序列如SEQ ID NO.:8所示。The porcine O-type FMDV bifunctional nanobody targeting porcine DC cells according to claim 5, wherein the amino acid sequence of the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells is as SEQ ID NO. :8 shown.
  10. 核酸或基因，其编码权利要求5所述的靶向猪DC细胞的猪O型FMDV双功能纳米抗体，其核苷酸序列如SEQ ID NO.:4所示。Nucleic acid or gene, the porcine O-type FMDV bifunctional nanobody of targeting porcine DC cell described in its coding claim 5, its nucleotide sequence is as shown in SEQ ID NO.:4.
  11. 权利要求1～9任一项所述的靶向猪DC细胞的猪O型FMDV双功能纳米抗体 的构建方法，其特征在于，所述构建方法包括如下步骤：将编码靶向猪DC细胞的猪O型FMDV双功能纳米抗体Nb131-104的基因插入pMECS载体，然后导入大肠杆菌WK6感受态细胞，获得重组菌；诱导重组菌表达目的蛋白，裂解重组菌后纯化获得双功能纳米抗体Nb131-104。The method for constructing the porcine O-type FMDV bifunctional nanobody targeting porcine DC cells according to any one of claims 1 to 9, characterized in that the construction method comprises the following steps: encoding the porcine DC cell-targeting porcine The gene of O-type FMDV bifunctional nanobody Nb131-104 was inserted into the pMECS vector, and then introduced into Escherichia coli WK6 competent cells to obtain recombinant bacteria; the recombinant bacteria were induced to express the target protein, and the bifunctional nanobody Nb131-104 was purified after lysing the recombinant bacteria.
  12. 靶向猪CD205的猪O型FMDV双功能纳米抗体，其特征在于，所述靶向猪CD205的猪O型FMDV双功能纳米抗体以接头元件将编码CD205目的蛋白特异性纳米抗体Nb193的基因和编码O型FMDV抗原特异性纳米抗体Nb104的基因连接后获得目的基因片段进一步重组表达获得的目的蛋白。The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 is characterized in that the porcine O-type FMDV bifunctional nanobody targeting porcine CD205 uses a linker element to encode the gene encoding the CD205 target protein-specific nanobody Nb193 and encode After the gene connection of the O-type FMDV antigen-specific nanobody Nb104, the target gene fragment was obtained by further recombinant expression of the target protein.
  13. 根据权利要求12所述的靶向猪CD205的猪O型FMDV双功能纳米抗体，其特征在于，所述猪CD205目的蛋白特异性纳米抗体Nb193的核苷酸序列如SEQ ID NO.:9所示，所述猪O型FMDV抗原特异性纳米抗体Nb104的核苷酸序列如SEQ ID NO.:2所示。The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 according to claim 12, wherein the nucleotide sequence of the porcine CD205 target protein-specific nanobody Nb193 is as shown in SEQ ID NO.:9 , the nucleotide sequence of the porcine O-type FMDV antigen-specific Nanobody Nb104 is shown in SEQ ID NO.:2.
  14. 根据权利要求12所述的靶向猪CD205的猪O型FMDV双功能纳米抗体，其特征在于，所述猪CD205目的蛋白特异性纳米抗体Nb193的氨基酸序列如SEQ ID NO.:11所示，所述猪O型FMDV抗原特异性纳米抗体Nb104的氨基酸序列如SEQ ID NO.:6所示。The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 according to claim 12, wherein the amino acid sequence of the porcine CD205 target protein-specific nanobody Nb193 is as shown in SEQ ID NO.: 11, wherein The amino acid sequence of the porcine O-type FMDV antigen-specific nanobody Nb104 is shown in SEQ ID NO.:6.
  15. 根据权利要求12所述的靶向猪CD205的猪O型FMDV双功能纳米抗体，其特征在于，所述接头元件为接头元件(G4S)4，所述接头元件(G4S)4的核苷酸序列如SEQ ID NO.:3所示，接头元件(G4S)4的氨基酸序列如SEQ ID NO.:7所示。The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 according to claim 12, wherein the linker element is a linker element (G4S) 4, and the nucleotide sequence of the linker element (G4S) 4 As shown in SEQ ID NO.:3, the amino acid sequence of linker element (G4S) 4 is shown in SEQ ID NO.:7.
  16. 根据权利要求12所述的靶向猪CD205的猪O型FMDV双功能纳米抗体，其特征在于，所述靶向猪CD205的猪O型FMDV双功能纳米抗体的氨基酸序列如SEQ ID NO.:12所示。The porcine O-type FMDV bifunctional nanobody targeting porcine CD205 according to claim 12, wherein the amino acid sequence of the porcine O-type FMDV bifunctional nanobody targeting porcine CD205 is as SEQ ID NO.: 12 shown.
  17. 核酸或基因，其编码权利要求16所述的靶向猪CD205的猪O型FMDV双功能纳米抗体，其核苷酸序列如SEQ ID NO.:10所示。Nucleic acid or gene, the porcine O type FMDV bifunctional nanobody of its coding target porcine CD205 described in claim 16, its nucleotide sequence is as shown in SEQ ID NO.:10.
  18. 权利要求12～16任一项所述的靶向猪CD205的猪O型FMDV双功能纳米抗体的制备方法，其特征在于，包括以下步骤：将双功能纳米抗体Nb193-104的编码基因插入pMECS载体，然后导入大肠杆菌WK6感受态细胞，获得重组菌；诱导重组菌表达目的蛋白，裂解重组菌后纯化获得双功能纳米抗体Nb193-104。The preparation method of the porcine O-type FMDV bifunctional nanobody targeting porcine CD205 according to any one of claims 12 to 16, characterized in that it comprises the following steps: inserting the coding gene of the bifunctional nanobody Nb193-104 into the pMECS vector , and then introduced into Escherichia coli WK6 competent cells to obtain recombinant bacteria; the recombinant bacteria were induced to express the target protein, and the recombinant bacteria were lysed and purified to obtain the bifunctional nanobody Nb193-104.
  19. 靶向猪CD205的猪PEDV双功能纳米抗体，其特征在于，所述靶向猪CD205的猪PEDV双功能纳米抗体以接头元件将编码CD205目的蛋白特异性纳米抗体Nb193和编码猪PEDV抗原特异性纳米抗体Nb2的基因连接后获得获得目的基因片段进一步重组表达获得的目的蛋白。The porcine PEDV bifunctional nanobody targeting porcine CD205 is characterized in that the porcine PEDV bifunctional nanobody targeting porcine CD205 uses a linker element to encode CD205 target protein-specific nanobody Nb193 and porcine PEDV antigen-specific nanobody After the gene connection of antibody Nb2 is obtained, the target gene fragment is further recombinantly expressed to obtain the target protein.
  20. 根据权利要求19所述的靶向猪CD205的猪PEDV双功能纳米抗体，其特征在于，所述猪CD205目的蛋白特异性纳米抗体Nb193的核苷酸序列如SEQ ID NO.:9所示，所述猪PEDV抗原特异性纳米抗体Nb2的核苷酸序列如SEQ ID NO.:13所示。The porcine PEDV bifunctional nanobody targeting porcine CD205 according to claim 19, wherein the nucleotide sequence of the porcine CD205 target protein-specific nanobody Nb193 is as shown in SEQ ID NO.: 9, wherein The nucleotide sequence of the porcine PEDV antigen-specific nanobody Nb2 is shown in SEQ ID NO.:13.
  21. 根据权利要求19所述的靶向猪CD205的猪PEDV双功能纳米抗体，其特征在于，所述猪CD205目的蛋白特异性纳米抗体Nb193的氨基酸序列如SEQ ID NO.:11所示，猪PEDV抗原特异性纳米抗体Nb2的氨基酸序列如SEQ ID NO.:14所示。The porcine PEDV bifunctional nanobody targeting porcine CD205 according to claim 19, wherein the amino acid sequence of the porcine CD205 target protein-specific nanobody Nb193 is as shown in SEQ ID NO.: 11, porcine PEDV antigen The amino acid sequence of the specific Nanobody Nb2 is shown in SEQ ID NO.:14.
  22. 根据权利要求19所述的靶向猪CD205的猪PEDV双功能纳米抗体，其特征在于，所述接头元件为接头元件(G4S)4，所述接头元件(G4S)4的核苷酸序列如SEQ ID NO.:3所示，接头元件(G4S)4的氨基酸序列如SEQ ID NO.:7所示。The porcine PEDV bifunctional nanobody targeting porcine CD205 according to claim 19, wherein the linker element is a linker element (G4S) 4, and the nucleotide sequence of the linker element (G4S) 4 is as SEQ As shown in ID NO.:3, the amino acid sequence of linker element (G4S) 4 is shown in SEQ ID NO.:7.
  23. 根据权利要求19所述的靶向猪CD205的猪PEDV双功能纳米抗体，其特征在于，所述靶向猪CD205的猪PEDV双功能纳米抗体的氨基酸序列如SEQ ID NO.:16所 示。The pig PEDV bifunctional nanobody targeting pig CD205 according to claim 19, wherein the amino acid sequence of the pig PEDV bifunctional nanobody targeting pig CD205 is as shown in SEQ ID NO.:16.
  24. 核酸或基因，其编码权利要求19所述的靶向猪CD205的猪PEDV双功能纳米抗体，其核苷酸序列如SEQ ID NO.:15所示。Nucleic acid or gene, which encodes the porcine PEDV bifunctional nanobody targeting porcine CD205 according to claim 19, its nucleotide sequence is as shown in SEQ ID NO.:15.
  25. 权利要求18～22任一项所述的靶向猪CD205的猪PEDV双功能纳米抗体的制备方法，其特征在于，包括以下步骤：将双功能纳米抗体Nb193-2的编码基因插入pMECS载体，然后导入大肠杆菌WK6感受态细胞，获得重组菌；诱导重组菌表达目的蛋白，裂解重组菌后纯化获得双功能纳米抗体Nb193-2。The preparation method of the porcine PEDV bifunctional nanobody targeting porcine CD205 according to any one of claims 18 to 22, characterized in that it comprises the following steps: inserting the coding gene of the bifunctional nanobody Nb193-2 into the pMECS vector, and then Introduce Escherichia coli WK6 competent cells to obtain recombinant bacteria; induce recombinant bacteria to express the target protein, lyse the recombinant bacteria and purify to obtain bifunctional nanobody Nb193-2.
  26. 权利要求1～9任一项所述的靶向猪DC细胞的猪O型FMDV的双功能纳米抗体、权利要求12～16任一项所述的靶向猪CD205的猪O型FMDV双功能纳米抗体、权利要求19～23任一项所述的靶向猪CD205的猪PEDV双功能纳米抗体、权利要求10、17或24所述的核酸或基因在制备猪用疫苗中的应用。The bifunctional nanobody of porcine O-type FMDV targeting porcine DC cells according to any one of claims 1 to 9, and the porcine O-type FMDV bifunctional nanobody targeting porcine CD205 described in any one of claims 12 to 16 Application of the antibody, the porcine PEDV bifunctional nanobody targeting porcine CD205 according to any one of claims 19 to 23, the nucleic acid or gene according to claim 10, 17 or 24 in the preparation of porcine vaccines.
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